IL224966A - Lyophilized therapeutic peptibody formulations - Google Patents

Lyophilized therapeutic peptibody formulations

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Publication number
IL224966A
IL224966A IL224966A IL22496613A IL224966A IL 224966 A IL224966 A IL 224966A IL 224966 A IL224966 A IL 224966A IL 22496613 A IL22496613 A IL 22496613A IL 224966 A IL224966 A IL 224966A
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IL
Israel
Prior art keywords
protein
peptide
peptides
formulation
formulations
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IL224966A
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Hebrew (he)
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Amgen Inc
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Application filed by Amgen Inc filed Critical Amgen Inc
Publication of IL224966A publication Critical patent/IL224966A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
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    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Description

LYOPHTLIZED THERAPEUTIC FORMULATIONS FIELD OF THE INVENTION the invention relates to formulations oflyophilized therapeutic peptibodies and methods for making a composition comprising therapeutic The present application is a divisional application of IL BACKGROUND OF THE INVENTION J Recombinant proteins are an emerging class of therapeutic Such recombinant therapeutics have engendered advances in protein formulation and chemical Modifications have been identified that can protect therapeutic primarily by blocking their exposure to proteolytic Protein modifications may also increase the therapeutic circulation and biological A review article describing protein modification and fusion proteins is Francis Focus on Growth Factors One useful modification is combination of a polypeptide with an domain of an Antibodies comprise two functionally independent a variable domain known as which binds and a constant domain known as which links to such effector functions as complement activation and attack by phagocytic An Fc has a long serum whereas an Fab is Capon et Nature See Patent When constructed together with a therapeutic or Fc domain can provide longer or incorporate such functions as Fc receptor protein A complement fixation and perhaps even placental Table summarizes use of Fc fusions with therapeutic proteins known in the Fc fusion with therapeutic proteins Form Fc Fusion Therapeutic implications Reference l Patent anaplastic leukemia Murine a Zheng et transplant rejection TN septic shock Van ei receptor autoimmune or disorders issued September the first IgGl CD4 receptor AIDS Capon el f Nature IgGl antiviral et 1 IgGl of WO published OPG density July of filed December 1 1 Human autoimmune disorders Polyethylene glycol conjugated or fusion proteins and peptides have also been studied for use in on artificial and other applications where biocompatibility is of Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to pharmaceuticals and implants and to molecules and surfaces For PEG derivatives have been proposed for coupling PEG to surfaces to control static and attachment of other types of molecules to the including proteins or protein in coupling of PEG has been shown to be desirable in overcoming obstacles encountered in clinical of biologically active Published PCT Publication WO for that many potentially therapeutic proteins have been found to have a short half life in blood PEGylation decreases the rate of clearance from the bloodstream by increasing the apparent molecular weight of the Up to a certain the of glomerular filtration of proteins is inversely proportional to the size of The ability of to decrease is generally not a function of how many groups are attached to the but the overall molecular weight of the conjugated Decreased clearance can lead to increased efficacy over the for Conforti et Research 287 and in PEGylation can decrease protein et Acta 248 alter protein immunogeniciiy et 252 3582 and increase protein solubility as for in PC Publication WO j J In the interaction of a protein ligand with its receptor often takes place at a relatively large as demonstrated in the ease of human growth hormone bound to its only a key residues at the interface actually contribute to most of the binding et Science This observation and the fact that the bulk of the remaining protein ligand serves only to display the binding epitopes in the right topology makes it possible to active ligands of much smaller molecules of only length as defined herein can bind to the receptor protein of a given large protein Such peptides may mimic the bioactivity of the large protein ligand through competitive inhibit the bioactivity of the large protein ligand Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and for Scott et ah Science Devlin et Science issued issued 31 issued issued Juh 1 i issued issued Juh WO published and WO published In such random peptide sequences are displayed by fusion with coat proteins of filamentous the displayed peptides are against an immobilized extracellular domain of a The retained phages may be enriched by successive rounds of affinity purification and and the best binding peptides are sequenced to identify residues one or more structurally related families of Cwirla el Science 276 which two distinct families were The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DN A Mutagenesis libraries may be created and screened to further optimize the sequence of the best Struct Other methods compete with phage display in peptide A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in Another method allows display on the outer membrane by fusion with a lipoprotein These and related methods are collectively referred to as coli Another biological approach to screening soluble peptide mixtures uses yeast for expression and See Smith et The method of Smith et al and related methods are referred to as In another translation of random RNA is halted prior to ribosome resulting in a library of polypeptides with their associated RNA still This and related methods are referred to as di Other methods employ chemical linkage of peptides to Roberts Szostak This and related methods are collectively referred to as Chemically derived peptide libraries have been developed in which peptides immobilized on such as polyethylene rods or Another chemically derived peptide library uses photolithography to peptides immobilized on glass These and related methods are collectively referred to as screening may be advantageous in that it use of acids and other unnatural as well as Both biological and chemical methods are reviewed in Wells Lowman 4 Ϊ j In the case of known bioactive rational design peptide ligands with favorable therapeutic properties can be carried In such an stepwise changes are made to a peptide sequence and the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide solution is These techniques are collectively referred to as In one such a series of peptides is made in which a single residue at a time is replaced with This technique is commonly referred to as an an When two residues or spaced are it is referred to as a alanine The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic Structural analysis of interaction may also be used to suggest peptides that mimic binding activity of large protein In such an the crystal structure may suggest the identity and relative orientation of critical residues of the large protein from which a peptide may be Takasaki et Nature These and related methods are referred to as structural These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage which may suggest further modification of the peptides to increase binding peptide mimetics of any protein can be identified using phage display and the other methods mentioned These methods have also been used for epitope for of critical a ino acids in and as leads for tiie discovery new therapeutic Cortese el Opin Peptide libraries are now being used most often in immunological such as epitope The Scientist Of particular interest is use of peptide libraries and other techniques in the discovery of pharmacologically active A number of such peptides identified in the art are summarized in Table The peptides are described in the listed The pharmacologic activity of the peptides is and in many instances is followed by a shorthand term therefor in Some of these peptides have modified to form peptide libraries were screened for binding to a receptor for a pharmacologically active protein at one instance the peptide library was screened for binding to a monoclonal Table 2 Pharmacologically active peptides Binding Form of Pharmacologic Reference peptide protein of activity interest1 intrapeptide EPO receptor Science bonded June el receptor cl Science dimer Wrighton e Nature Biotechnology International patent application WO published 1996 linear EPO receptor Naranda et WO published September 1999 Science stimulation of hematopoiesis Paukovits et linked dimer Physiol The protein listed in this column may be bound by the associated peptide EPO or mimicked by associated The references listed for each clarify whether the molecule is bound by or mimicked by the Shatnagar et linked dimer on et King et Blood 86 309a linear receptor inflammatory and autoimmune diseases or et Acad et 271 et Yanofsky linear Facteur stimulation of lymphocytes et al scrique Cellular Yoshida Int CTIA4 et disulfide Nature bonded cxocyclic receptor antagonist Takasaki et Nature WO published December receptor antagonist trapcpiide C3b inhibition of complement cl disulfide autoimmune bonded diseases Protein linear adhesion processes Alley et ceil wound tumor metastasis linear C4 binding et protein linear urokinase receptor processes associated with et urokinase interaction with its receptor International tumor cell invasion and application WO published October 1997 linear Hdm2 Inhibition of inactivation of Picksicy et p53 mediated by Mdm2 or Oncogene et Boitger ct Oncogene linear by mimicking Ball et activity o linear by preventing Gibbs ct Cell transferase activation of oncogene 178 linear Ras effector by inhibiting et biological function of the rns Trends Genet oncogene Rodriguez et Nature linear by inhibiting Pawson el al domains tumor growth with activated tyrosine treatment Yu of disease Rickies cl states Sparks Sparks cl i US issued March US issued March 1999 by mimicking el activity of pi inhibiting complex linear of Mast cell activation I hypersensitivity ceil linear protease treatment of inflammatory International application disorders mediated by WO published release of 1998 protease linear antigen treatment of viral Dyson Moray infections linear selcciins Martens 1 inflammatory diseases Biol European patent application EP published June 96 calmodulin ct Diversity I Adey Kay treatment for International conditions related to WO published cellular June WO including platelet published March WO wound published tissue WO published angiogenesis May Kraft treatment of and invasion linear and treatment of inflammatory WO published extracellular and autoimmune conditions March 98 components off cells and macrophages linear somatostatin and treatment or prevention of European patent application 0 1 published April gastric tumor inhibition of hormone modulation of sleep or neural activity bacterial septic disorders by issued March linear or mellitin antipathogenic WO published 28 August 1997 including acids cyclic WO published disorders October linear CTLs cancer 0 770 published May linear linear Cooper linear rheumatoid diabetic psoriasis cyclic inflammation and Nature autoimmune tumor growth fragment treatment of obesity Pat Echistatin inhibition of platelet Gan aggregation linear WO published October alpha suppression of metastasis FEBS 44 I endothelial cell activation Blank et atuiphospholipid syndrome USA thromboembolic antibodies and recurrent loss linear Cell Receptor diabetes WO published chain April 19 antiviral January growth WO published December WO published December linear Apoptosis treatment WO published ofT disorders autoimmune viral cell T cell class treatment of autoimmune US diseases issued March androgen useful in WO published treating cancer September hunlingtm linear von inhibition of factor 1 WO published Fuel or anticoagulants April linear lcnli virus CLP I antimicrobial US issued linear sleep disorders Graf Inducing Peptide 1 linear and cancer Cancer Protein r infertility Suzuki Peptides Bioche angiotensins hematopoietic factors for conditions Periodontal from linear linear inhibition of bone resorption linear antimicrobial Ilarvig Methods 1 AH Park linear antagonist WO published linear oilier diseases WO published proteins linear envelope treatment of neurological WO published protein degenerative diseases linear autoimmune disorders published graft rheumatoid WO published March Peptides fled by peptide library screening have been regarded as in of therapeutic agents rather than being used as therapeutic agents Like other proteins and they would be rapidly removed in vivo either by renal cellular clearance mechanisms in the reticuloendothelial or proteolytic Focus on Growth Factors 1 As a the art presently uses the identified peptides to validate drug targets or as scaffolds for design organic compounds that might not have been as easily or as quickly identified through chemical library Lowman Kay Drug Today purified peptides are marginally stable in an aqueous state and undergo chemical and physical degradation resulting in a loss of biological activity during processing and peptide compositions in aqueous solution undergo such as deamidation and peptide bond These effects represent a serious problem for active peptides which are intended to be administered to humans within a defined dosage range based on biological Administration of purified peptides remains a promising treatment strategy for many diseases that affect the human the ability of the therapeutic to remain a stable pharmaceutical composition over time in a variety of storage conditions and then be effective patients in has not been there remains a in the art to provide therapeutic peptibodies in stable formulations that are useful as therapeutic agents for the treatment of diseases and SUMMARY OF THE INVENTION The present invention provides formulations useful for of therapeutic resulting in a highly stable therapeutic The stable therapeutic peptibody product is useful as a therapeutic agent in the of individuals suffering from disorders or conditions that benefit from the administration of the one the invention provides a lyophilized therapeutic peptibody composition comprising a a bulking a stabilizing and optionally a wherein the buffer is comprised of a pH buffering agent in a range of about 5 mM to about and wherein the pH is in a range of about 3 0 to about wherein the bulking agent is at a concentration of about to about wherein the stabilizing agent is at a concentration of about to about the surfactant is at a of about to about and wherein therapeutic peptibody comprises structure set out in Formula F1 is an selected from X is selected wherein and are each independently sequences of pharmacologically active and L5 each independently and h are each independently or 1 provided that at least one of a and b is 1 d is I or greater than and WSP is a water soluble the attachment of which is effected at any reactive moiety in In another embodiment the therapeutic peptibody comprises a structure set out in Formula 11 Formula wherein Fc domain is attached at the of and one or more WSP is attached to the Fc optionally through linker still another the therapeutic peptibody comprises a structure set out in Formula wherein the Fc domain is attached at the of and one or more WSP is attached to the Fc optionally through linker In yet another the therapeutic peptibody comprises a structure set out in Formula IV Formula 1 wherein the Fc domain is attached at the of and one or more WSP is attached to the Fc optionally through linker In another the therapeutic peptibody comprises a structure set out in Formula V Formula wherein the Fc domain is attached at the and one or more WSP is attached to the Fc optionally through linker In another the therapeutic peptibody is a multimcr or In another an aforementioned composition is provided wherein P3 P4 are independently selected from a peptide set out in any one of Tables 4 through In a related embodi P3 P4 have the same amino acid sequence in another the Fc domain is out in SEQ ID 1 another WSP is In still another the domain is et out in SEQ ID and WSP is In another the PEG has a molecular weight of between about 2 kDa and or between kDa and 25 In another embodi the composition comprises at least or therapeutic yet another embodiment of the an aforementioned composition is provided wherein the pH buffering agent selected from the group consisting of and In yet another embodiment of aforementioned composition is provided wherein the bulking agent selected from the group consisting of or In yet another embodiment of the an aforementioned composition is provided wherein the stabilizing agent selected from the group consisting of arginine polysaccharides such as cellulose and hyaluronic sodium In yet another embodiment of an aforementioned composition is provided wherein the surfactant selected from the group consisting of sodium lauryl dioctyl sodium dioctyl sodium chenodeoxycholic lauroylsarcosine sodium lithium 1 acid sodium sodium cholale sodium acid sodium bcnzalkonium chloride or benzethonium chloride Triton Triton lauromacrogol polyoxyl 40 polyoxyethylene hydrogenated castor oil 50 and glycerol polysorbate 65 and soy DM and sucrose fatty acid methyl cellulose and yet another embodiment of the an aforementioned composition is provided wherein the therapeutic peptibody concentration is between about and 250 in another embodiment of the an aforementioned composition is provided wherein the pH buffering agent is histidine and wherein the pH is wherein the bulking agent is wherein the stabilizing agent is and wherein the surfactant is In another the aforementioned composition is provided wherein P1 comprises a sequence set forth in Table In yet another embodiment an aforementioned composition is provided wherein the therapeutic peptibody concentration is In another the therapeutic peptibody is any one of SEQ ID ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 101 1 SEQ SEQ ID SEQ ID In yet another embodiment an aforementioned composition is provided wherein the pH buffering agent is histidine and wherein the pH is wherein the bulking agent is wherein the stabilizing agent is and wherein the surfactant is in another aforementioned composition is provided wherein comprises a sequence set forth in Table in yet another embodiment of the an aforementioned composition is provided wherein the therapeutic peptibody concentration is 30 In still another embodiment of the an aforementioned composition is provided wherein the buffering agent is 20 mM histidine and wherein the pH is wherein the bulking agent is wherein the stabilizing agent is and wherein the surfactant is another the aforementioned composition is provided wherein comprises a sequence set forth in Table in yet another embodiment of the an aforementioned composition is provided wherein the therapeutic peptibody concentration is still another embodiment the an aforementioned composition is provided wherein the pH buffering agent is mM histidine and wherein the pH is wherei the bulking agent is and wherein the stabilizing agent is In another the aforementioned composition is provided wherein comprises a set forth in Table in yet another embodiment of the an aforementioned composition is provided the therapeutic peptibody concentration is In another embodiment of the aforementioned composition is provided wherein the composition is from the group consisting pH mannitol and with and without mM pH mannitol and with and without M pH mannitol and sucrose with and without mM pH mannitol and mM pH mM pH 1 5 mM pH and pH In another the aforementioned composition is provided wherein comprises a sequence set forth in Tables still another the aforementioned composition is provided wherein the therapeutic peptibody concentration is selected from the group consisting of and The present invention also contemplates methods of making lyophiiized therapeutic peptibodies of the present in one the invention provides a method for making a therapeutic peptibody comprising steps preparing a solution of a a bulking a stabilizing and a optionally wherein the buffer is comprised of a pH buffering agent in a range of about 5 mM to about 20 mM and wherein the pH is in a range of about to about wherein the bulking agent is at a concentration of about to about wherein the stabilizing agent is at a concentration of about to about wherein the surfactant is at a concentration of about to about and the therapeutic wherein the therapeutic peptibody comprises a structure set out in Formula Formula wherein is an is selected from X is selected wherein and arc each sequences of pharmacologically active and each independently and h are each 0 or I provided that at least one of a and h is 1 d is or greater than 1 and WSP is a water soluble the attachment of which is effected at any reactive moiety in another the aforementioned method is provided wherein thee therapeutic peptibody comprises a structure set out in Formula wherein the Fc domain is attached at the of and one or more WSP Is attached to the Fc optionally through linker In another the aforementioned method is provided wherein the therapeutic peptibody comprises a structure set out in Formula wherein the Fc domain is attached at the of and one or more WSP is attached to the Fc optionally through linker In another the aforementioned method is provided wherein the therapeutic peptibody comprises a structure out in Formula IV Formula wherein the Fc domain is attached at the P1 and one or more WSP is attached to the Fc optionally through linker in another the method is provided the therapeutic peptibody comprises a structure set out in Formula V Formula wherein the Fc domain is attached at the N of and one or more WSP is attached to the Fc optionally through linker in another aforementioned method is provided wherein the therapeutic peptibody is a multi er or In another the P4 are independently selected from a peptide set out in any one oG Tables 4 through another the the same amino acid In another the Fc domain is set out in SEQ ID another WSP is In another the Fc domain is out in SEQ 1 and WSP is In another PEG has a molecular weight of between about 2 kDa and 100 kDa or between about 6 kDa and 25 in another the method is provided wherein the composition comprises at least or PEGylated therapeutic In another the aforementioned method is provided wherein the pH buffering agent is selected from the group consisting of and another the aforementioned method is provided wherein the bulking agent selected from the group consisting of or In another the aforementioned method is provided wherein the stabilizing agent selected from the group consisting of glucose arginine including polysaccharides such as methyl cellulose and hyaluronic sodium another the aforementioned method is provided wherein the surfactant selected from the group consisting of sodium dioctyl sodium sodium chenodeoxycbolic sodium lithium dodecyl acid sodium sodium cholate sodium acid sodium chloride or benzelhonium chloride hexadecyltrimelbylammonium Triton Triton polyoxyl 40 polyoxyethylene hydrogenated castor oil 50 glycerol polysorhate 65 soy DM and sucrose fatty acid methyl cellulose and carboxymcthyl In another the aforementioned method is provided wherein the therapeutic peptibody concentration is between about and 250 j another an method is provided wherein the pH buffering agent is histidine and wherein the pH is wherein the bulking agent is wherein stabilizing agent is and wherein the surfactant is another the aforementioned method is provided wherein comprises a sequence set forth in another the aforementioned method is provided wherein the therapeutic peptibody concentration is j In another an aforementioned method is provided wherein the pH buffering agent is mM histidine and wherein the pH is wherein the bulking agent is wherein the stabilizing agent is and wherein the surfactant is in another the aforementioned method is provided wherein P1 comprises a sequence set forth Table another the aforementioned method is provided wherein the therapeutic peptibody concentration is 30 in another an aforementioned method is provided wherein the pH buffering agent is 20 histidine and wherein the pH is wherein the bulking agent is wherein the stabilizing agent is and wherein the surfactant is another the aforementioned method is provided wherein P1 comprises a sequence forth in Table In another the orementioned method is provided wherein the therapeutic peptibody concentration is another an aforementioned method is provided wherein the pH buffering agent is 10 mM histidine and wherein the pH is wherein the bulking agent is and wherein stabilizing agent is In another 22 i the aforementioned method is provided comprises a sequence set forth in Table In another the aforementioned method is provided the therapeutic peptibody concentration is 30 another embodiment of the an method is provided wherein the composition is selected from the group consisting mM pH mannitol and with and without pH mannitol and with and without mM pH mannitol and sucrose with and without mM pH 4 mannitol and mM pH mM pH hydroxyethyl 5 mM pH and 10 mM pH another the aforementioned method is provided wherein comprises a sequence set forth in Tables 21 In still another the aforementioned method is provided wherein the therapeutic peptibody concentration is selected from the group consisting of and In another an aforementioned method is provided further prior to the steps adjusting the pH of the solution to a pH between about and about preparing a solution containing the therapeutic buffer exchanging the solution of step into the solution of step adding an appropriate amount of a and lyophiiizing the mixture step in another the aforementioned method Is provided wherein a method for preparing a reconstituted therapeutic peptibody composition is provided comprising the steps lyophiiizing an aforementioned therapeutic peptibody and reconstituting the therapeutic peptibody In another a kit for preparing an aqueous pharmaceutical composition is provided comprising a first container having an aforementioned therapeutic peptibody and a second container having a physiologically acceptable solvent for the lyophilized DETAILED DESCRIPTION OF THE INVENTION Definition of terms The term with respect to a peptide means a compound may include additional amino acids on either or both of the a or carboxy termini of the given Of course these additional amino acids should interfere with activity of the With respect to a composition of the instant the term means that a composition may include additional These additional components should not significantly interfere with the activity of the The term refers to a molecule comprising fused either directly or indirectly to other molecules such as an domain of an where the peptide moiety specifically binds to a desired The may be fused to either Fc region or inserted into an a modified fc are described in Patent Application Publication The invention such molecules comprising an Fc domain modified to comprise a peptide as an internal sequence in a loop of the Fc The Fc internal peptide molecules may more than one peptide sequence in tandem in a particular internal and they may include further peptides in other internal While the putative loop regions are insertions in any other domains of the Fc are also considered part of this The term does not include proteins full length proteins fused to an Fc The term refers to a molecule that prevents degradation increases reduces reduces immunogenici or increases biological activity of a therapeutic Exemplary vehicles include an Fc domain as described in Patent to Capon et issued The term refers to molecule or sequence comprising the sequence of a fragment resulting from digestion of whole whether in monomeric or multimeric a native Fc comprises a and CM3 The original immunoglobulin source of the native Fc is in one aspect of human origin and may be any of the A native Fc is a monomeric polypeptide that may be linked into dimeric or multimeric forms by covalent association disulfide covalent association or a combination of The number of disulfide bonds between monomeric subunits of native Fc molecules ranges from one to four depending on class or subclass One example of a native Fc is dimer resulting from digestion of an Ellison et Nucleic Acids The term as used is generic to the and The term refers to a molecule or sequence that is modified from a native but still comprises a binding site for the salvage International applications WO 25 September and WO describe exemplary Fc as well as interaction with the salvage In one the term comprises a molecule or sequence that is humanized from a another a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present term comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in disulfide bond incompatibility with a selected host cell heterogeneity upon expression in a selected host interaction with binding to an Fc receptor other than a salvage or cellular cytotoxicity Fc variants are described in further detail The term encompasses native Fc and Fc variant molecules and sequences as defined As with Fc variants and native the term includes molecules in monomeric or multimeric whether digested from whole antibody or produced by other In one for the Fc region can YKTTPPVLDSDGSFFLYSKLTVD ID Additional Fc sequences are known in the art and are contemplated for use in the For Fc IgG Accession No Fc IgG2 Accession Fc Accession Fc lgG4 Accession Fc Accession Fc lgA2 Accession No Fc Accession Fc Accession and IgE Accession are some additional Fc sequences contemplated for use an amino acid sequence may added to the above sequences where expressed in The term as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated or by both covalent and molecules typically form and or Multimers may be formed by exploiting the sequence and resulting activity of the native source of the Fc or by derivatizing defined such a native Tire terms or mean processes and resulting compounds in for example and without the compound has a cyclic for between residues within the the compound is or has a for compound has a cysteinyl residue and thus forms dimers in culture or in or more linkage is replaced by a the is replaced by a succinitnide or substituted or unsubslituled wherein R and and the ring substituents are as defined the is replaced by wherein and are as defined and compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal Derivatives are further described As used herein the term refers to molecules of 1 13 14 21 31 38 71 81 91 100 or more amino acids linked by peptide Peptides typically contain random flexible such as random and typically lack stable such as those observed in larger typically via secondary and tertiary In particular numerous size ranges of peptides are contemplated such from 26 amino acids in and the further the peptides used herein are no more than or 20 amino acids in Exemplary may be generated by any of the methods set forth such as carried in a peptide library a phage display generated by chemical derived by digestion of or generated using recombinant DNA Peptides include D and either purified or in a mixture of the two physiologically acceptable salts of compounds of this invention are also By acceptable is meant any salts that are known or later discovered to be pharmaceutically Some specific examples such as hydrochloride and and The term as used to refer to peptide sequences refers to fully random sequences selected by phage display and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring Exemplary methods for identifying peptide sequences include phage E ribosome chemical rational protein structural and the The term means that a substance described is determined to have activity that affects a medical parameter but not limited to blood blood ceil cholesterol or disease but not limited to autoimmune pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined The terms and refer a peptide having biological activity comparable to a protein but not limited to and other proteins described that interacts with a protein of These terms further include peptides that indirectly mimic the activity of a protein of such as by potentiating the effects of the natural ligand of the protein of for the peptides listed in Tables 2 and As an the term comprises any peptides that can be identified or derived as described in Wrighton Science Naranda et USA or any other reference Table 2 identified as having subject Those 27 L of ordinary skill in the art appreciate that each of these references one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide j As another the term or refers to peptides that can be identified or derived as described in Science and and any other reference in Table 2 identified as having subject as well as International application WO published May Those of ordinary skill in the art appreciate that each of references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries As another the term refers to any peptides that can be identified or described in Paukoviis el or any of the references in Table 2 identified as having subject Those of ordinary skill in the art appreciate that each olThese references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different pepti e The term refers to any peptides that can be identified or derived as described in Fukumoto et Nature Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide The terra or refers to a peptide that blocks or in some way interferes with the biological activity of the associated protein of has biological activity comparable to a known antagonist or inhibitor of the associated protein of the term comprises peptides that can be identified or derived as described in Takasaki ct Nature Biotech or any of the references in Table 2 identified as having T subject Those of ordinary skill the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide The terms I and refers to peptides that inhibit or activation receptor by l receptor activation results from formation of a complex among and receptor accessory 1 antagonist or peptides hind to or receptor accessory protein and obstruct complex formation among any two or three components of the Exemplary antagonist or can identified or derived as described in or any of the references in Table 2 identified as having l or antagonistic subject Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides actually disclosed therein by following the disclosed procedures different peptide The term refers to peptides that can be identified or derived as described in and in any of the references in Table 2 identified as having subject Those of ordinary skill in the art appreciate each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide The term inhibitor refers to peptides that can be identified or derived as described in Nature and in any of the references in Table 2 identified as MMP inhibitory subject Those of ordinary skill in the appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with peptide The term inhibitor refers to peptides that can be identified by their to reduce or block activity or signaling as demonstrated in in vitro assays such for example the pM ARE myostatin activity assay or by in vivo animal testing as described patent application Publication No A and PCT application publication Exemplary myostatin inhibitor peptides are set out in Tables The term refers to peptides that inhibit or regulate activity of cell adhesion integrin selectin or cell adhesion molecule Exemplary antagonists comprise the peptides described in Tables The term resorption refers to such molecules as determined by the assays of Examples 4 and 1 1 of WO Exemplary bone inhibitors OPG and which are described in WO and The term growth factor or growth factor refers to a peptide that binds to and inhibits nerve growth factor activity or Exemplary peptides of this type arc set out in Table The term modulating refers to any amino acid sequence that binds to the and comprises naturally occurring sequences or randomized sequences Exemplary 1 modulating domains can be identified or derived by phage display or other methods mentioned Exemplary peptides of this type are set out in Tables and The term refers to a molecule that hinds to the and increases or decreases one or more assay parameters opposite from the effect on those parameters by full length native Such activity can be for by such assays as described in the subsection entitled activity of in the Materials Methods section of the patent application WO published August The term refers to peptides that can be identified or derived as having Exemplary peptides of this type are set out in Tables The term refers to a water soluble polymer which prevents a protein or other compound to which it is attached from precipitating in aqueous such by way of a physiological A more detailed description of various WSP embodiments contemplated by the invention Administration Therapeutic are useful pharmaceutical formulations in order to treat human diseases as described one embodiment the therapeutic peptibody compositions are Lyophilization is out using techniques common in the art and should be optimized for the composition being developed et 21 and Chang et Pharm A lyophilization cycle in one of primary and secondary drying Phil Trans R Soc Ser Biol In the freezing the solution is cooled to initiate ice this slop induces the crystallization of the bulking The ice sublimes the primary which is conducted by reducing chamber pressure below the vapor pressure of the using a vacuum and introducing heat to promote adsorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and at an elevated The process produces a material known as a iyophilized Thereafter the cake can be reconstituted with either sterile or suitable diluent for j The lyophilization cycle not only determines the final physical state of but also affects other parameters such as reconstitution stability and final moisture The composition structure in the frozen state proceeds through several transitions glass and that occur at specific temperatures and can be used to understand and optimize the lyophilization The glass transition temperature can provide information about the physical slate of a solute and be determined by differential scanning calorimetry Tg and are an important parameter that must be taken into account when designing the lyophilization For is important for primary in dried the glass transition temperature provides information on the storage temperature of the final Excipients in general Excipients are additives that are included in a formulation because either imparl or enhance the delivery and manufacturability of a drug Regardless of the reason for their excipients are an integral component of a drug product and therefore need to be safe and tolerated by For protein choice of excipients is particularly important because they can affect both efficacy and tmmunogcnicity of the protein formulations need to be developed with appropriate selection of excipients that afford suitable and A iyophilized formulation is usually comprised of a a bulking and a utility of a surfactant may be evaluated and selected in cases where aggregation during the lyophilization step or during reconstitution becomes an An appropriate buffering agent is included to maintain the formulation within stable zones of pH during A comparison of the excipient components in liquid and lyophilized protein formulations is provided Table Excipient components of lyophilized protein formulations The principal challenge in developing formulations for therapeutic proteins is stabilizing the product against the stresses of shipping and The role of formulation excipients is to provide stabilization against these Excipients may also be employed to reduce viscosity high concentration protei formulations in order to enable their delivery and enhance patient excipients can be classified on the basis of the mechanisms by which they tabilize proteins against various chemical and physical Some excipients are used to alleviate the effects of a specific stress or to regulate a particular of a specific Other excipients have more general effects on the physical and covalent stabilities of The excipients described herein are organized either by their chemical type or functional role in Brief descriptions of the modes of stabilization are provided when discussing each Given the teachings and guidance provided those skilled in the art will know what amount or range of excipient can be included in any particular formulation to achieve a biopharmaceutical formulation of the invention that promotes retention in of the biophar For amount and type of a salt to be included in a biopharmaceutical formulation of the invention can be selected based on to the desired osmolality hypotonic or of the final solution as well as the amounts and osmolality of other components to be included in the by exemplification with reference to the type of polyol or sugar included in a the amount of such an excipient will depend on its By way of inclusion ofabout sorbitol can achieve isotonieity while about o a sucrose excipient is needed to achieve Selection of the amount or range of concentrations of one or more excipients that can be included within a biopharmaceutical formulation of the invention has been exemplified above by reference to polyols and those skilled in art will understand that the considerations described and further by reference to specific excipients are equally applicable to all types and combinations of excipients for amino other tonicity bulking metal chelating agents where a particular excipient is reported a formulation percent those skilled in the art will recognize that the equivalent molar concentration of that excipient is also Of a person having ordinary skill in the art would recognize that the concentrations of the aforementioned excipients share an interdependency within a particular By way of the concentration of a bulking agent may be lowered there is a high concentration or there is a high stabilizing agent a person having ordinary skill in the art would recognize in order to maintain the isoionicity of a particular formulation in which there is no bulking the concentration of a stabilizing agent would be adjusLed accordingly a amount of stabilizer would be Other excipients are known in the art and can be found in et Compendium of Excipients fir Parenteral Formulations PDA Buffers j The stability of a protein drug is usually observed to be maximal in a narrow pH This pH range of optimal stability needs to be identified early during formulation Several approaches such as accelerated stability studies and calorimetric screening studies have been demonstrated to be useful in this endeavor et Once a formulation is the drug product must be manufactured and maintained within a predefined specification throughout its are almost always employed to control pH in the Organic phosphates and Tris have employed routinely as buffers in protein formulations The buffer capacity of the buffering species is maximal at a pH equal to the pKa decreases as pH increases or decreases away from this Ninety percent of the buffering capacity exists within one pH unit of its Buffer capacity also increases proportionally with increasing buffer Several factors need to be considered when choosing a First and Lhe buffer species and its concentration need to be defined based on its and the desired formulation Equally important is to ensure that the buffer is compatible with the protein other formulation and does not catalyze any degradation polyanionic carboxylate bufers such as citrate and succinate have been shown to form covalent adducts with the side chain residues of A third important aspect to be considered is the sensation of stinging and irritation the buffer may For citrate is known to cause stinging upon injection cl Basic Clin Pharmacol Toxicol The potential for stinging and irritation is greater for drugs that are administered via the SC or where the drug solution remains at the site for a relatively longer period of time than when administered by the route where the formulation gets diluted rapidly into the blood For formulations that are administered by direct the total amount of buffer other formulation needs to be One has to be particularly careful about potassium ions administered in the form of the potassium phosphate which can induce cardiovascular effects in a patient et Physician Buffers for formulations need additional Some buffers like sodium phosphate can crystallize out of protein amorphous phase during freezing resulting in rather large in Other common buffers such as acetate and imidazole should be avoided since they may sublime or evaporate during the thereby shifting the pH of formulation during lyophilization or after Table Commonly used buffering agents and their values The buffer system present in the compositions is to be physiologically compatible and to maintain a desired pH in the reconstituted solution as well as in the solution before In one the pH of the solution prior to is between pH and pH For in one embodiment pH of the solution prior to is 1 1 1 1 or In another the pH of the reconstituted solution is between and In one the pH in the reconstituted solution is or In one the pH buffering agent used in the formulation is an amino acid or mixture of amino In one the pH buffering agent is histidine or a mixture of amino acids one of which is The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined in one when the pH buffering agent is an amino the concentration of the amino acid is between mM and one the pH buffering agent is at least 1 1 or 900 another the concentration of pH buffering agent is between or 90 mM and still another the concentration of the pH agent is between or 40 mM and 50 yet another the concentration of the pH bufferi g agent is j0098J Other exemplary pH buffering agents used to buffer the formulation as set out herein but are not limited to and Stabilizers and bulking agents Bulking agents are typically used in formulations to enhance product elegance and to prevent Conditions in the formulation are generally designed so that the bulking agent crystallizes out of the frozen amorphous phase during freezing or annealing above the giving the cake structure and Mannitol and glycine are examples of commonly used bulking j include a class of compounds that can serve as cryo and glass forming Cryoproteclants act to stabilize proteins during freezing or in the frozen state at low temperatures Good Pharmaceutical Buffalo Lyoprotectants stabilize proteins in the solid dosage form by preserving the conformational properties of the prolcin during dehydration of Glassy state properties have been lied as or depending on their relaxation properties as a function of It is important that and glass forming agents remain in the same phase with the protein in order to impart and polyols fall into this category can sometimes serve all three Polyols encompass a class excipients that includes and other polyhydric alcohols glycerol and propylene The polymer polyethylene glycol is included in this Polyols are commonly used as stabilizing excipients isotonicity agents in both liquid and parenteral protein With respect to the the polyols are kosmolropic and are preferentially excluded from the protein Polyols can protect proteins from both physical and chemical degradation Preferentially excluded increase the effective surface tension of solvent at the protein interface whereby the most energetically favorable protein conformations are those with the smallest surface Mannitol is a popular bulking agent in Iyophilized formulations because it crystallizes out of the amorphous protein phase during lending structural stability to the cake Leukine Enbrel is generally used in combination with a cryo lyoprolectant like Because of the propensity of mannitol to crystallize under frozen sorbitol and sucrose are the preferred tonicity in liquid formulations to protect the product against stresses encountered during transport or when freezing bulk prior to Sorbitol and sucrose are far more resistant to and therefore less to phase separate from the protein is interesting to note that mannitol has been used in tonici lying amounts several marketed liquid formulations such as and the product labels of these drugs Not The use of reducing sugars free aldehyde or ketone such as glucose and lactose should be avoided because they can react glycate surface lysine and arginine residues of proteins via the Hard reaction of aldehydes and primary amines et Humeny ct Agric Food Sucrose can hydrolyze to fructose and glucose under acidic conditions and Robinson and consequently may cause The polymer polyethylene glycol could stabilize proteins by two different temperature dependent At lower it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperature given its nature and Lee Biochemistry Thus at lower temperatures it may protect proteins via the mechanism of preferential but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules PEG is also a cryoprotectant and has been employed in a formulation of recombinant Antihemophilic which utilizes PEG 3350 at a concentration of The low molecular weight liquid 300 600 can be contaminated with peroxides and cause protein If the peroxide content in the raw material must be minimized and controlled throughout its The same holds true for In a particular embodiment of present a stabilizer a combination of is added to formulation to prevent or reduce or aggregation and chemical A hazy or turbid solution upon reconstitution indicates that the protein has The term means an excipient capable of preventing aggregation or other physical as well as chemical degradation in an aqueous and solid Stabilizers that are conventionally employed tn pharmaceutical compositions but are not limited arginine including polysaccharides such as methyl cellulose and hyaluronic sodium et Standard in one the stabilizer is incorporated in a concentration of about to about In another the stabilizer is incorporated in a concentration of at least 1 or another the stabilizer is incorporated in a concentration of about to about In still another tire stabilizer is incorporated in a concentration of about to about yet another the stabilizer is incorporated in a concentration of about the compositions also include appropriate amounts of bulking and osmolarity regulating agents suitable for forming a Bulking agents may be either crystalline or amorphous polymers such as Other exemplary bulking agents include or In one the bulking agent is In a further the bulking agent is incorporated in a concentration of about to about in another the bulking agent is incorporated a concentration of at least 0 1 or In a yet further embodiment in a concentration of about to to produce a mechanically and pharmaceutically stable and elegant In another the mannitol concentration is Surfactants Protein molecules have a high propensity to interact with surfaces making them susceptible to adsorption and at and This degradation pathway has been observed to be inversely dependent on protein concentration and result in either the formation of soluble and insoluble protein aggregates or the loss of protein from solution via adsorption to surfaces addition to container surface degradation is exacerbated with physical as would be experienced during shipping and handling of the Surfactants are commonly used in protein formulations to prevent induced Surfactants are amphipathic molecules with the capability of competing proteins for interfacial Hydrophobic portions of the surfactant molecules occupy positions while hydrophilic portions of the molecules remain oriented towards the bulk At sufficient concentrations around the critical micellar a surface layer of surfactant serve to prevent protein molecules adsorbing at the degradation is The most commonly used surfactants are fatty acid esters of and HO The two di fer only in the length of aliphatic chain that imparts hydrophobic character to and is more and has a lower critical micellar concentration than The surfactant poloxamer has also been used in several marketed liquid products such Norditropin and Ovi Detergents can also affect the thermodynamic conformational stability of the effects of a given excipient will be protein For polysorbates have been shown to reduce the stability of proteins and increase the of Detergent destabilization proteins can be rationalized in terms of the hydrophobic tails of the detergent molecules that can engage in specific binding with partially or wholly unfolded protein These types of interactions could cause a shift in the conformational equilibrium towards the more expanded protein states increasing the exposure of hydrophobic portions of the protein molecule in complement to binding if the protein native state exhibits some hydrophobic detergent binding to the native state may stabilize that I Another aspect of polysorbates is that they are inherently susceptible to oxidative as raw they contain sufficient quantities of peroxides to cause oxidation of protein residue especially The potential for oxidative damage arising from the addition of stabilizer emphasizes the point that lowest effective concentrations of excipients should be in For the effective concentration for a given protein will depend on the mechanism of has been postulated that if the mechanism of surfactant stabilization is related to preventing on the effective concentration will be around critical micellar if the mechanism of stabilization is associated specific the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction el Pharm Surfactants may also be added in appropriate to prevent surface related aggregation phenomenon during freezing and drying Exemplary surfactants include and amphoteric surfactants including surfactants derived from amino Anionic surfactants but are not limited sodium lauryl dioctyl sodium sulfosuccinate and dioctyl sodium sodium lithium dodecyl acid sodium sodium sodium glycodeoxycholie acid sodium Cationic surfactants but arc not limited chloride or benzethonium cetylpyridinium chloride and bromide Zwitterionic surfactants but arc not limited and surfactants but are not limited Triton Triton and In another surfactants include polyoxyl 40 polyoxyethylene hydrogenated castor oil 50 and glycerol polysorbate 65 and soy lecithin and other phospholipids such as DM DM and sucrose fatty acid methyl cellulose and Compositions comprising these either individually or as a mixture in different are therefore further In one the surfactant is incorporated in a concentration of about to about in another is incorporated in a concentration of at least 1 or In another the surfactant is incorporated in a concentration of about to about In still another the surfactant is incorporated a concentration or to about yet another the surfactant is incorporated in a concentration of about to about Salts Salts often added to increase the ionic strength of the which can important for protein physical and Salts can affect the physical stability of proteins in a variety of Ions can stabilize the native state of proteins by binding to charged residues on the they can stabilize the denatured stEile by binding to the peptide groups along the protein backbone can also stabilize the protein native conformation by shielding repulsive electrostatic interactions between residues within protein Electrolytes in protein formulations can also shield attractive electrostatic interactions between protein molecules that can lead to protein aggregation and effect of salt on the stability and solubility of proteins varies significantly with the characteristics of the ionic Hofmeister series originated in the as a way to rank order electrolytes based on their ability to precipitate proteins et Quarterly Reviews of Biophysics 241 in this the Hofmeister scries is used to illustrate a scale of protein stabilization effects by ionic and co in Table are ordered with to their general effects on solution state from stabilizing to destabilizing in the differences in effects across the anions are far greater than that observed f the for both the effects are most apparent at higher concentrations than are parenteral concentrations of kos o tropes molar ammonium are commonly used to precipitate proteins from solution by a process called where the is preferentially excluded from the protein surface reducing the solubility of the protein in native Removal or dilution of the salt will return the protein to The term refers to the use of destabilizing ions guanidine and that increase the solubility of proteins by solvating the peptide bonds of the protein Increasing concentrations of the will favor the denatured stale conformation of the protein as the solubility of the peptide chain The relative effectiveness of ions to and defines their position in the Hofmeister In order to maintain isotonicity in a parenteral salt concentrations are generally limited to less than for monovalent ion In this concentration the mechanism of salt stabilization is probably due to screening of electrostatic repulsive intramolecular forces or attractive forces Huckcl chaotropic salts have been shown to be more effective at stabilizing the protein structure than similar concentrations of kosmotropes by this The chaotropic are believed to bind more strongly than the With respect to covalent protein differential effects of ionic strength on this mechanism are expected through published reports of protein stabilization by sodium chloride are accompanied by those where sodium chloride accelerated covalent mechanisms by which salts affect protein stability are protein and may vary significantly as a function of solution pH An example where an excipient can useful in enabling the delivery of a protein drug is that high concentration antibody salts have shown to be effective in reducing the viscosity of such formulations Erratum Pharm j Amino acids Amino acids found versatile use in protein formulations as bulking stabilizers and Histidine and glutamic acid are employed to buffer protein formulations in the pH range of and The imidazole group of histidine has a and the carboxyl group of glutamic acid side chain has a pKa of which makes suitable for buffering in their respective pH most commonly used buffer in the acidic pH range of sublimates during and hence should not be used in Glutamic acid is particularly useful in such cases is commonly found in marketed protein formulations It provides a good alternative to a buffer known to sling upon injection histidine has also been reported to have a stabilizing effect on with respect to aggregation when used at high concentrations in both liquid and lyophilized presentations et Pharm Histidine to 60 was also observed to reduce the viscosity of a high concentration formulation of this in the same the authors observed increased aggregation and discoloration in histidine containing formulations during studies of the antibody in stainless The authors attributed this to an effect of iron ions leached from corrosion of steel Another note of caution with histidine is that it undergoes in the presence of metal ions el Biochemistry The use of as an in formulations appears it has been observed to be effective against a number of oxidative stresses et Pharm Sci 5 The amino acids serine and alanine have been shown to stabilize proteins by the mechanism of preferential Glycine is also a commonly used bulking agent in lyophilized formulations a crystallizes out of the frozen amorphous phase giving the cake structure and Arginine has been shown to be an effective agent in inhibiting aggregation and has been used in both liquid and lyophilized formulations the enhanced efficiency of refolding of certain proteins in the presence of arginine has been attributed to its suppression of the competing aggregation reaction during Antioxidants Oxidation of protein residues arises from a number of different Beyond the addition of specific the prevention of oxidative protein damage involves the careful control number of factors throughout the manufacturing process and storage of the product such as atmospheric light and chemical The most commonly used pharmaceutical antioxidants are reducing chelating Antioxidants in therapeutic protein formulations must be and remain active throughout the product Reducing agents and scavengers work by ablating active oxygen species in Chelating agents such as EDTA can be effective by binding trace metal contaminants that promote For EDTA was utilized in the liquid formulation of acidic fibroblast growth factor to inhibit the metal catalyzed oxidation of cysteine EDTA has in marketed products like and addition to evaluating the effectiveness of various excipients at preventing protein formulation scientists must be aware of the potential for the antioxidants themselves to induce other covalent or physical changes to the A number of such eases have been reported i the Reducing agents can cause disruption of intramolecular disulfide which can lead to disulfide In the presence of transition metal ascorbic acid and EDTA have been shown to promote methionine oxidation in a number of proteins and peptides and Defelippis Peptides and Proteins as Parenteral Pharmaceutical Formulation Development of Peptides and Sven Lars Pharmaceutical Taylor and UK Fransson Pharm Yin el Pharm 21 Sodium thiosulfate has been reported to reduce the levels of light and temperature induced in rhuMab the formation of a adduct was also reported in this study Yang et J Pharm Sci Selection of an appropriate antioxidant is made according to the specific stresses and sensitivities of the Metal ions In transition metal ions are undesired in protein formulations because they can physical and chemical degradation reactions in specific metal ions are included in formulations when they are to proteins and in suspension formulations of proteins where they form coordination complexes zinc suspension of the use of magnesium ions has been proposed to inhibit the isomerization of aspartic acid to Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease and Factor the case of ions to increased the stability of the enzyme through a specific binding site el Pharm In removal of calcium ions from the solution with caused an increase in deamidation and thi effect was observed with other divalent cations and were observed to destabilize Similar effects were observed in Factor Ca12 and ions stabilized the protein while others like and Zn 2 and destabilized the enzyme et In a separate study with Factor a significant increase in aggregation rate was observed in the presence of et Pharm Sci authors note that other excipients like buffer are often contaminated with A ions and illustrate the need to use excipients of appropriate quality in formulated Preservatives Preservatives are necessary when developing parenteral formulations that involve more than one extraction from the same Their primary function is to inhibit microbial growth and ensure product sterility throughout the or term of use of the drug Commonly used preservatives include benzyl phenol and Although preservatives have a long history the development of protein formulations that includes preservatives can be Preservatives almost always have a destabilizing effect on and this has become a major factor in limiting their use in protein formulations et 96 To most protein drugs have been formulated for when formulations are they have the added advantage of enabling patient and increased A good example that of human growth hormone where the development of preserved formulations has led to commercialization of more injection pen At least four such pen devices containing preserved formulations of hGH arc currently available on the Novo Nutropin Genotropin dual chamber Pharmacia contain phenol while is formulated with Several aspects need to be considered during the formulation development of preserved dosage The effective preservative concentration in the drug product be This requires testing a given preservative the dosage form with concentration ranges that confer effectiveness without compromising protein For example three preservatives were successfully screened in the development of a liquid formulation for receptor using differential scanning calorimetry The preservati es were rank ordered based on their impact on stability at concentrations commonly used in marketed products Jr et As might be development of liquid formulations containing preservatives are more challenging than products can without the preservative and reconstituted with a preservative containing diluent at the time of This shortens the for which a preservative is in contact with protein significantly minimizing the associated stability With liquid preservative effectiveness and stability have to be maintained over the entire product life An important to note is that preservative effectiveness has to be demonstrated in the final formulation containing the active drug and excipient Some preservatives can cause injection site which is another factor that needs consideration when a clinical trials that focused on the evaluation of preservatives and buffers In Nordi pain perception was observed to be lower formulations containing phenol and benzyl alcohol as compared to a formulation containing 62 among the commonly used benzyl alcohol possesses anesthetic properties and Sun Given the teachings and guidance provided those skilled in the art will know what amount or range of excipient can be included any particular formulation to achieve a formulation of the invention that promotes retention in stability of the For the amount and type of a salt to be included in a biopharmaceutical formulation of the invention can be selected based on to the desired osmolality hypotonic or of the final solution as well as the amounts and osmolality of other components to be included in the by exemplification with reference to the type of polyol or sugar included a the amount of such an excipient will depend on By way of inclusion of about sorbitol can achieve isotonicity while about of a sucrose excipient is to achieve Selection of the amount or range of concentrations of one or more excipients that can be included within a formulation of the invention has above by reference to polyols and those skilled in the art will understand that the considerations described herein and further exemplified by reference to specific excipients are equally applicable to all types and combinations of excipients for amino other tonicity bulking metal chelating agents where a particular excipient is reported in a formulation percent those skilled in the art will recognize that the equivalent molar concentration of that excipient is also j Of a person having ordinary skill in the art would recognize that the concentrations of the aforementioned excipients share an interdependency within a particular By way of concentration of a bulking agent may be lowered there is a high concentration or there is a high stabilizing agent in a person having ordinary skill in the art would recognize in order to maintain the isotonicity of a particular formulation in which there is no bulking the concentration of a stabilizing agent would adjusted accordingly a amount of stabilizer would be The compositions are stable for at least two years at to in the This stability is beneficial for extending the shelf of the pharmaceutical Methods of Preparation The present invention further contemplates methods for the preparation of therapeutic protein formulations one methods for preparing a therapeutic formulation comprising the step of a therapeutic peptibody in a buffer comprising a buffering a bulking a stabilizing agent and a The present methods further comprise one or more of the following adding a stabilizing agent to said mixture prior to adding at least one agent selected from a hulking an regulating and a surfactant to said prior to The bulking agent may be any bulking agent described one the bulking agent is another the stabilizing agent is The surfactant may be any surfactant described one the surfactant is The standard reconstitution practice for lyophilized material is to add back a volume of water or water for injection equivalent to the volume removed during although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration Drug Development and Industrial methods are provided for preparation of reconstituted therapeutic peptibodies comprising the step of adding a diluent to a lyophilized therapeutic peplibody composition of the j The lyophilized therapeutic peptibody composition may be reconstituted as an aqueous A variety of aqueous sterile water for water with preservatives for multi dose or water with appropriate amounts of surfactants or aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous various such excipients are suspending for example sodium sodium gum and gum dispersing or wetting agents may be a for example or condensation products of an oxide with fatty for example polyoxyethylene or condensation products of ethylene oxide with long aliphatic for example or condensation products of ethylene oxide with partial esters derived fatty acids and a hexitol such as polyoxyethylene sorbitol or condensation products of ethylene oxide with partial esters derived from fatly acids and hexitol for example polyethylene The aqueous suspensions may also contain one or more for example or To administer compositions to human or test in one the compositions comprises or acceptable The phrases or refer molecular entities and compositions that are inhibit protein degradation such aggregation and cleavage and in addition do not produce or other adverse reactions when administered using routes in the as described acceptable include any and all clinically useful dispersion antibacterial and antifungal isotonic and absorption delaying agents and including those agents disclosed The therapeutic compositions may be administered by inhalation rectal I or by intracranial The term parenteral as used herein includes subcutaneous nal or infusion Administration by intrapulmonary injection and or surgical implantation at a particular site is contemplated as compositions are essentially free of as well as other impurities that could be harmful to the Single or multiple administrations of the compositions can be carried out with the dose levels and pattern being selected by the treating For the prevention or treatment of the appropriate dosage will depend on the type of disease to be as defined the severity and course of the whether drug is for preventive or therapeutic previous clinical history and response to the and the discretion of attending As an additional the invention includes kits which comprise one or more compounds or compositions packaged in a manner which facilitates their use for administration to In one such a includes a compound or composition described herein a composition comprising a therapeutic protein or packaged in a container such as a seeded bottle or with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the In one the kit contains a first container having a therapeutic protein or peptide composition and a second container having a physiologically acceptable reconstitution solution for In one the compound or composition is packaged in a unit dosage The kil may Further include a device suitable for administering the composition according to a fic route of the kit contains a label that describes use of therapeutic protein or peptide Dosages The dosage regimen involved a method for treating a condition described herein will be determined by the attending considering various factors which the action of the body sex and diet of the the severity of any time of administration and other clinical In various the daily regimen is in range of of a preparation per kilogram of body weight the mass of the protein without chemical or in some embodiments of the the dose may exceed 3 or Preparations of the invention may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug As another the inventive compound may be administered as a Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient The frequency dosing depend on the parameters of the agents and the route of The pharmaceutical formulation be determined by one skilled in the art depending upon the route of administration and desired See for Pharmaceutical Mack Publishing PA pages Such formulations may influence the physical rate of in and rate of in vivo clearance of the administered Depending on the route of a suitable dose may be calculated according to body body surface area or organ Further refinement of the calculations necessary to determine the appropriate dosage for treatment invol ing each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue especially in light of the dosage information and assays disclosed as as the pharmacokinetic data observed in the human clinical trials discussed Appropriate dosages may be ascertained through use of established assays for blood level dosages in r conjunction with appropriate The final dosage regimen will be 51 determined by the attending considering various factors which modify the action of the specific the severity the damage and the responsiveness of the the body sex and diet of the the severity of any of administration and other clinical As studies are further information emerge regarding the appropriate dosage levels and duration of treatment for various diseases and i Structure of compounds in General in preparations in accordance with the a peptide is attached to a vehicle through the of the of the or and the resulting structure may be further modified with a covalently attached WSP which is attached to the vehicle moiety in the the therapeutic molecules of this invention may be described by the following formula I 1 is a X is selected from X2 selected wherein and P4 are each independent sequences of pharmacologically active and arc each independently and h are each independently or provided that at least of arid b is i is or greater than 1 and WSP is a soluble the attachment of which is effected at any reactive moiety in Ϊ compound comprises compounds of formulae ii including multimcrs wherein is an Fc domain and is attached at the of and one or more WSP is attached to the Fc optionally through linker including multimcrs wherein is an Fc domain and is attached at the of and one or WSP is attached to the Fc optionally through linker IV including multimcrs wherein is an Fc domain is attached at the of one or more WSP is attached to the Fc optionally through linker wherein F1 is an Fc domain and attached at the and one or WSP is attached to the Fc optionally through linker In one is an Fc domain and is attached to either the or of a a related the Fc is linked into a dimeric form as described herein to which 2 peptides arc The peptides may be homodimeric the same amino acid or hetcrodynammic different amino acid sequences that bind the same target or that bi d j another comprising a are Loops comprising a are prepared a process in which at least one biologically active peptide is incorporated as an internal sequence into an Fc Such an internal sequence may be added by insertion between amino in the previously existing Fc or by replacement of amino acids the previously existing Fc domain removing amino acids in the previously existing Fc domain and adding peptide amino the latter the number of peptide amino acids added need not correspond to the number of amino acids removed from the previously existing Fc For in onc a molecule in which amino acids are removed and amino acids are added is Pharmacologically active compounds provided are prepared by a process selecting at least one peptide that modulates the activity of a protein of and preparing a pharmacologic agent comprising an amino acid sequence of the selected peptide as an internal sequence of an Fc This process may be employed to modify an Fc domain that is already linked through an or or sidechain to a as described in and and international publication numbers WO and WO The process described in Patent Application Publication may also be employed to modify an Fc domain that is part of an In this different molecules can be produced that have additional such as a binding domain to a different epitope or an additional binding to the precursor existing Molecules comprising an internal peptide sequence are also referred to as internal or internal peptide The Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal and they may include further peptides in other internal While the putative loop regions are insertions in any other domains of the Fc are also considered part of this Variants and derivatives of the above compounds are also encompassed by this The compounds of this invention may he prepared by standard synthetic recombinant or other methods of preparing peptides and fusion A use contemplated for Fc internal peptide molecules is as a therapeutic or a prophylactic A peptide may have activity comparable even greater natural ligand mimicked by the in certain natural therapeutic agents might induce antibodies against the own endogenous the unique sequence of the peptide avoids this pitfall by having little or typically no sequence identity with the natural the Fc internal peptibodies may have advantages in refolding and purification over or linked Fc Further Fc internal peptibodies may be more stable in both due to the stabilization of chimeric and due to increased to prolcolytic degradation from and Fc internal peptibodies may also exhibit improved pharmacokinetic Peptides Any number peptides may used in with the present Of particular interest are peptides that the activity growth and Peptide antagonists are also of particularly those antagonistic to the activity of any of the interleukins and proteins involved in complement activation Targeting peptides arc also of including transporting and the All of these classes of peptides may be discovered by methods described in the references cited in this specification and other Phage in is useful in generating peptides for use in the present It has stated that affinity selection from libraries of random peptides can be used to y peptide ligands for any site of any gene et Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as surface receptors or any proteins having linear Wilson el Kay et Drug Today Such proteins are extensively reviewed in et Receptor Signal Transduction Such proteins of interest are preferred for use in this By way of example and a group of peptides that bind to cytokine receptors are Cytokines have recently been classified according to their receptor See Archivum et Therapiae Among these receptors are the CKRs 1 in The receptor classification appears in Table Table 3 Cytokine Receptors Classified by Receptor Code 7R belongs lo is unassigned 4 indicated subj ami Other type subtypes remain Hematopoietic ligands and not possess functional intrinsic protein The signaling molecules cytokines arc STATs and related been cloned bul according to the receptor code they remain receptors use distinct molecules lor signal transduction including of R and 55 kDa that participates in their cytotoxic R bind relntetl factors as well receptors seven domain They arc G i j Other proteins of interest as targets for peptide generation present invention include the The group is receptor that can bind several R belongs to the immunoglobulin but their signal transduction events characteristics remain neurotrophic cytokines associate with receptors may encompass many other factors remain protein kinases are intrinsic part of the intracellular domain of receptor kinase The participate In the signals transmission via the anb3 B7 Calcitonin CD28 CETP factor B C4b CTLA4 Glucagon Glucagon Receptor splice variants of molecules preferentially expressed on tumor CD30 unglycosylated variants of mucin and Lewis Y surface glycoproleims CD CD45 prostate specific membrane antigen and prostate specific cell matrix both secreted and receptor urokinase plasminogen activator UFA receptor parathyroid hormone parathyroid protein RII Nerve growth factor and receptors Selectins receptors thereof Cell adhesion molecules and receptors thereof Exemplary appear in Tables 4 through 38 These peptides may be prepared by any methods disclosed in the many of which are discussed In most tables that single letter amino acid abbreviations are The in these sequences throughout this speci unless specified otherwise a particular means that any of the naturally occurring amino acid residues may be Any of these peptides may be linked in tandem with or without a few examples are provided in the Linkers are listed as and may be any of the linkers described Tandem repeats and are shown separated by dashes for Any peptide containing a residue may be with another either or both of which may be linked to a A few examples are provided in the Any peptide having more than one Cys residue may form an intrapeptide disulfide as for peptides in Table A few examples of intrapeptide peptides are specified in the Any of these peptides may be derivalized as described and a few derivatized examples are provided in the Derivatized peptides in the tables are rather than as the associated peptides may he employed in this as For derivatives in which the carboxyl terminus may be capped with an amino the capping amino group is shown as For derivatives in which amino acid residues arc substituted by moieties other than amino acid the substitutions are denoted by which signifies any of the moieties described in Bhatnagar el and Cuthbertson et The substituent and the substituents are as defined in Pat and For the sequences the substituents X2 through and the integer are as defined in WO Also for the the substituents follow the definitions and of WO The substituents and as defined in Sparks et are as defined in Pat except for and are as defined applications published June 1995 and WO published March and for mimetic X and Z and the integers m and n are as defined WO published October Xaa and below are as defined in WO published March and AC are as defined in application WO published December and X4 in Table only are as defined in European application EP 0 91 1 published April Residues appearing in boldface are All peptides are linked through peptide bonds unless otherwise Abbreviations are listed at end of this In the ID means that sequence listing required for the given Table 4 peptide sequences 62 5 peptide sequences Table 6 peptide sequences Tabic 7 peptide sequences Table 8 peptide sequences Tabic 9 peptide Table 10 Selectin antagonist peptide sequences peptide sequences Tabic 12 8 Ta peptide sequences Table 14 Calmodulin antagonist sequences Table 15 Mast cell cell protease inhibitor peptide sequences antagonist peptide sequences Table Somatostatin or cortistatin mimetic peptide sequences Table antagonist sequences 88 L inhibiting peptide sequences 20 Additional Exemplary Pharmacologically Active Peptides TABLE PEPTIDES 95 1 96 j r TABLE 22 M YOST A TIN INHIBITOR PEPTIDES TABLE 23 IN INHIBITOR PEPTIDES 24 MYOSTATIN INHIBITOR PEPTIDES ί Tabic 25 sequences Table 26 Scicctin antagonist peptide sequences Table 27 binding peptides Table 28 peptide sequences 29 Modulating Peptides TALL MODULATING PEPTIDES T able 31 inhibitory 14 TABLE 32 INHIBITOR PEPTIDES TABLE 33 INHIBITOR PEPTIDES TABLE 34 INHIBITOR PEPTIDES i TABLE 35 INHIBITOR PEPTIDES 36 Binding Peptides 1 37 Binding Peptides Table 38 Binding Peptides j In addition to compounds set out in the invention provides numerous other compounds in one compounds comprise the following general TMP 2 wherein TMP and are each independently selected from group of compounds comprising core wherein X2 is selected from the group consisting of and X3 is from the group consisting of Gly and X4 is X5 is selected from the group consisting of Thr and is selected from the group consisting of and is selected from the group consisting of Arg and Xg is selected from the group consisting of and X9 is selected from the group consisting of and X is selected from the group consisting of and is a linker as described and n is 0 or and physiologically acceptable salts In one comprises wherein n is 1 through and when n is greater than up to half of the Gly residues may be substituted by another amino acid selected from the remaining natural amino acids or a stereoisomer In addition to the core structure above and other structures are also wherein one or more the following is added to the 2 core is attached to the X X are attached to the wherein X X and 4 are as is selected from the group consisting of and is selected from the group consisting of selected from the group consisting of and 3 is selected from the group consisting of and and is selected from the group consisting of and TMP compounds of the invention are made up at least subunits wherein the The subunits are amino acids independently selected from among the 20 amino the invention embraces compounds where independently selected from the group of occurring acids well known in the Specific amino acids are identified for each For 2 may be or Both and single letter abbreviations for amino acids are used each the abbreviations are the standard ones used for the amino acids or variations These amino acids may have either or D stereochemistry for which is neither nor and the TMPs well as all other compounds of the may comprise a combination of The invention also provides reverse TMP molecules well as other peptides disclosed wherein the amino terminal to terminal sequence of the amino acids is For the reverse of a molecule having the normal sequence X would be The invention also provides molecules well as for all other molecules of the invention described like a reverse the amino to terminal sequence of amino acids is reversed and residues that are normally enatiomers in TMP are altered to the stereoisomer Exemplary TMP compounds of the invention therefore include without limitation the following ID ID ID ID ID ID ID EG PTLRQ A AR A GG GN GSGG 1 EG PT LRQ W LA ARA ID ID ID ID F W A G 1 EG PT A A R A Fc ID ID ID ID S O U ID IEGPT LRQ AR GG EG PT LRQW LA ARA ID 129 I ID A Derivatives The invention also contemplates derivatizing the peptide an vehicle portion discussed of the Such derivatives may improve the biological half and the like of the The moieties may alternatively eliminate or attenuate any undesirable of the compounds and the Exemplary derivatives include compounds in The compound or some portion thereof is For the peptide portion may be modified to contain two or more Cys residues in the which could by bond For citations to references on preparation of cyclized sec Table The compound is or is rendered capable of between For the peptide portion may be to contain Cys residue and thereby be able to form an disulfide bond with a like The compound may also be through its as in the molecule shown One or more peptidyl linkages is replaced by pcptidyl Exemplary linkages are urea and alkylated peptide wherein is lower The is the may be or to a substituted Exemplary derivative groups include 1 than or wherein R and R are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C 1 C 1 and The free is the is or For one may use methods described in the art to add to compounds of this one may use methods described in the art to add to compounds of this Exemplary derivative groups for wherein R2 is lower alkoxy or wherein R3 and R4 are independently hydrogen or C alkyl C 1 A disulfide bond is replaced with preferably more linking moiety an Bhatnagar et Alberts et Thirteenth or more individual amino acid residues is modi Various derivatizing agents are known to react specifically with selected or terminal as described in detail Heterobifunctional polymers are typically used to link An example is or The NHS reacts with primary which upon conjugation at pH 7 is Once the complex is reaction of maldmide portion of can proceed with another containing a free which occurs at a much faster rate than formation of the amide in the initial The result is a link between two for An application is illustrated by the preparation fragments to horseradish peroxidase Yoshitake et lmagawa Uto et The use of Sulfo SMCC allows for water solubility so that an organic solubilization step is not allowing for greater flexibility and less disruption of activity reacting with Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid which reverse the charge of the lysinyl Other suitable reagents for derivalizing include imidoesters such as methyl pyridoxal and reaction with Arginyl residues may be modified by reaction with any one or combination of several conventional including and Derivatizalion of arginyl residues requires that the reaction be performed in alkaline conditions because of the high of the guanidine functional these reagents may read with the groups of lysine as well as the arginine Specific modification of residues has studied with particular interest in introducing spectral into tyrosyl residues by reaction with aromatic diazonium compounds or Most N imidizole and tetranitromethane are used to form tyrosyl species and Carboxyl sidechain groups or may be selectively modified by reaction with carbodi ides such as carbodiimide or 1 and glutamyl residues may be converted to asparaginyl and residues by reaction with ammonium Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl these residues are deamidated under mildly acidic Either form of these residues falls within the scope of this residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding to stabilize Bhatnagar et Deri atizalion with functional agents is useful for the peptides or their functional derivatives to a support matrix or to other Commonly used agents for with homobi including disuccinimidyl esters such as and bifunctional maleimides such as agents such as yield iates that are capable of forming crosslinks in the presence of matrices such as cyanogen carbohydrates and the reactive substrates described in and arc employed for protein Carbohydrate groups may conveniently be attached to sites that are known to be glycosylation sites in oligosaccharides are attached to serine or threonine residues while oligosaccharides are attached to asparagine residues when they are pari of the sequence where X can be any amino acid except X is preferably of the naturally occurring acids other than The structures of and go and the sugar residues found in each type are One type of sugar that is commonly found on both is acid to as sialic Sialic acid is usually the terminal residue of both and oligosaccharides by virtue of its negative may confer acidic properties to the glycosylated Such may be incorporated the linker of the compounds of this invention and are preferably glycosylated by a ceil during recombinant production of the polypeptide compounds in mammalian cells such as such sites may further be glycosylated by synthetic or procedures known in the Other possible modifications include hydroxylation of proline and phosphorylation of hydroxyl groups of seryl or threonyl oxidation of the sulfur atom in of the groups of and histidine side Structure and Molecule Properties Freeman San Compounds of the present Invention may be changed at the DNA as The DNA sequence oG any portion of the compound may be changed to codons more compatible with the chosen host For which is the preferred host optimized codons are known in the Codons may be substituted to restriction sites or to include silent restriction which may aid in processing of the DNA in the selected host The linker and peptide DNA sequences may be modified to include any of the sequence and derivatives Another set of useful derivatives are the molecules to or Such conjugation is especially useful for molecules comprising peptide that bind to tumor cells or Such molecules may be used as therapeutic agents or as an aid to surgery surgery or or as diagnostic agents or j As therapeutic these conjugated derivatives possess a number of They facilitate use of toxins and radioisotopes that would be toxic if administered without the specific binding provided by the peptide They also can reduce the effects that attend the use of radiaLion and chemotherapy by facilitating lower effective doses of the conjugation Useful conjugation partners such as and A derived toxins such as Pseudomonas endotoxin and the partner molecules in capture systems streptavidin as either partner molecules in capture systems or as especially for diagnostic and cytotoxic agents One useful adaptation of these conjugated derivatives is use in a capture system in such a the molecule of the present invention would comprise a benign capture This capture molecule would be able to bind to a separate effector molecule for a toxin or Both the conjugated molecule and the effector molecule would be administered to the patient in such a the effector molecule would have a short except when bound to the capture thus minimi ing any The conjugated molecule would have a relatively long but would be benign and The specific binding portions of both molecules can be part of a known specific binding pair or can result from peptide generation methods such as escribed Such conjugated derivatives may be prepared by methods known in the the case of protein effector molecules Pseudomonas such molecules can be expressed as fusion proteins from correlative DN A Radioisotope conjugated derivatives may be for as described for the BEXA antibody Derivatives comprising cytotoxic agents or microbial toxins may be for as described for the BR96 antibody Molecules employed in capture systems may be for as described by the patent and publications from Molecules employed for and RID may for by the patent and publications from Vehicles The invention requires presence of at least one vehicle attached to a peptide through or a sidechain of one of the amino acid vehicles may also be in one an Fc domain is the The domain may be fused to the N or C termini of the peptides or at both the N and C various embodiments of the the Fc component is cither a Fc or an Fc The immunoglobulin source of the native Fc in one of human origin and in alternative be of any class of Native Fc domains are made up of monomelic polypeptides that may be linked into dimeric or multimeric forms by covalent disulfide The number of disulfide bonds between monomeric subunits of native Fc ranges from one to four depending on class or subclass JgG 1 One example of a native Fc is a dimer resulting from papain digestion of an IgG Ellison el Nucleic Acids should be noted that Fc monomers will spontaneously dimerize when the appropriate cysteine residues are particular conditions are present that prevent dimerization through disulfide bond Even the cysteine residues that normally form disulfide bonds the Fc dimer are removed or replaced by other the monomeric chains will generally form a dimer through The term herein is used to any of these the native the native dimer bond modified dimers and modified monomers As Fc variants are suitable vehicles within the scope of this A native Fc may be extensively modified to form an Fc provided binding to the i salvage receptor is for example WO and WO In such Fc one may remove or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this One may remove these sites for substituting or deleting inserting residues into the or truncating portions containing the The inserted or substituted residues may also be altered amino such as pcptidomimetics or Fc variants may be desirable for a number of several of which are Exemplary Fc variants include molecules and sequences in Sites involved in disulfide formation are Such removal may avoid reaction with other proteins present in the host ceil used to produce the molecules of the For this the segment at the may be truncated or cysteine residues may be deleted or substituted other amino acids Even when cysteine residues are the single chain Fc domains can still form a dimeric Fc domain that is held together A native Fc is modified to make it more compatible with a selected host For one may remove the PA sequence near the of a typical native which may be recognized by a digestive enzyme in such as proline One may also add art methionine especially when the molecule is expressed recombinantly in a bacterial cell such as A portion of the of a native Fc is removed to prevent heterogeneity when expressed in a selected host For this one may delete any of the first amino acid residues at the those at positions 4 and One or more glycosylation sites are Residues that arc typically glycosylated may confer cytolytic Such residues may be deleted or substituted with unglycosyiated residues Sites involved in interaction with such as the Cl q binding are For one may delete or substitute the sequence of human IgG Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided such an Fc are removed that affect binding to Fc receptors other than a salvage A native Fc may have sites for interaction with blood cells that are not required for the fusion molecules of the present invention and so may be The ADCC site is ADCC sites are known in the for 29 with regard to ADCC sites in These as are not required for the fusion molecules of the present invention and so be When the native Fc is derived from a the native Fc may be to humanize a native one will substitute selected residues in the native Fc with residues that are normally found in human native Techniques for antibody humanization are known in the An alternative vehicle would be a antibody or small molecule a capable of binding to a salvage For one could use as a vehicle a polypeptide as described in Pat issued April to Presta et Peptides could also be selected by phage display for binding to the FcRn salvage Such salvage compounds are also included within the meaning of and are within the scope of this Such vehicles should be selected for increased by avoiding sequences recognized by and decreased by favoring immunogenic as discovered in antibody analogs or derivatives of the Fc portion may be constructed making various substitutions of residues or Variant polypeptides include insertion wherein one or more amino acid residues supplement an Fc acid Insertions may be located at either or both termini of the or may be positioned within internal regions of the Fc amino acid Insertion with additional residues at either or both can include for fusion proteins and proteins including amino acid tags or For the Fc molecule may optionally contain an especially when the molecule is expressed recombinanlly in a bacterial cell such as deletion one or more amino acid residues in an Fc polypeptide are Deletions can be effected at one or both termini of the Fc or with removal of one or more residues within the amino acid Deletion include all fragments of an Fc polypeptide In Fc substitution one or more amino acid residues of Fc polypeptide are removed and replaced with alternative residues one the substitutions are conservative in nature and conservative substitutions of this type are well known in the the invention embraces substitutions that are also Exemplary conservative substitutions are described in 2nd Worth York and set out immediately CONSERVATIVE I SIDE CHAIN CHARACTERISTIC ACID Aliphatic A L I V Aromatic F W M Borderline G Hydroxyl S T Y Amides N Q C Borderline G Positively charged R H Negatively charged D exemplary conservative substitutions are set out immediately CONSERVATIVE SUBSTITUTIONS ORIGINAL RESIDUE EXEMPLARY SUBSTITUTION Ala Asn Asn Arg Asp Asp His Arg Leu Phe Lys Asn Met Phe Ala Pro Gly Tlir Thr Ser Trp Tyr Tyr Ser Val Ala t ΐ For cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc Each cysteine residue can be removed substituted with other amino such as Ala or As another modifications may also be made to introduce amino acid substitutions to ablate the Fc receptor binding ablate the complement 1 binding to ablate the antibody dependent cytotoxicity Such sites are known in the and any known substitutions are within the scope of Fc as used For see Molecular with regard to ADCC sites in or more tyrosine residues can be replaced by phenylalanine In other variant amino acid deletions substitutions are also contemplated and are scope of the present Conservative amino acid substitutions will generally be alterations j may be in tire form of altered amino such as imetics or Fc sequences of cornpound may also be derivatized as described herein for bearing modifications other than or substitution of amino acid the modifications are covalent in and include for chemical bonding with other and inorganic Derivatives of the invention may be prepared to circulating or may designed to improve targeting capacity for the polypeptide to desired or It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the part of a compound of the such as described in WO entitled Polypeptides with Increased Additional members of the class of molecules designated as Fc herein are those that are described in WO entitled Domains with Increased WSP components Compounds of the invention may further include at least one The WPS moiety of the molecule may be branched or For therapeutic use of the product the polymer is pharmaceutically In a desired polymer is selected based on such considerations as whether the polymer conjugate will be used and the desired circulation resistance to and other various the average molecular weight of each water soluble polymer is between about 2 kDa and about between about 5 50 between about kDa and about 40 kDa and between about 20 kDa and about 5 In yet another aspect the molecular weight of each polymer is between about 6 kDa and about 25 The term as used herein and indicates that in preparations of a water soluble some molecules will weigh some than the stated molecular the higher the molecular weight or the more the higher the Other sizes may be depending on the desired therapeutic profile including for the duration of sustained the if on biological the case in the degree or lack of antigenicity and other known effects of a water soluble polymer on a therapeutic The WSP should be attached a polypeptide or peptide with consideration given to effects on functional or antigenic domains of the polypeptide or In chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer Activating groups which can be used to link the water soluble polymer to one or proteins without limitation oxirane and If attached to the peptide by reductive the polymer selected should have a single reactive aldehyde so that the degree of polymerization is clinically water soluble polymers include without glycol of ethylene polyvinyl alcohol polyvinyl anhydride poly homopolymers or random pyrrol propropylene glycol and other polyakylene polypropylene oxide polyols and other yethylated polyoxyethylateci or colonic acids or carbohydrate Ficoll and mixtures Water Soluble Polymers can also be made to be thermally as in the formation of reverse thermal Examples include with tetra armed and The hydrophilicity of polymers can be varied by substituting hydrophobic portions into the polymer An example of this is in the manufacture of which the ratio of lactic acid to glycolic acid can be increased to allow for lower water Lower water soluble polymers may be desired in certain for example in increasing the potential to interact with cell Upon an appropriate ratio of phospholipids may be used to induce the formation of micelles or liposomes in The advantage of such a may be in the ability to incorporate some of the protein within the with potential benefit of prolonging Phospholipids capable of forming liposomes or micelles include and apprpopriate secondary liposome strengthening components such as Certain such as phosphate and are capable of forming micelles in J Polysaccharide polymers are another type of water soluble polymer which may be used for protein or peptide Modifying proteins or peptides by adding may increase decrease increase stability and decrease Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by The dextran itself is available in many molecular weight and is readily available in molecular weights J from about kD to about 70 Dexlran is a suitable soluble polymer for use the present invention as a vehicle by itself or in combination with another vehicle for WO and WO The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has for European Patent Publication 0 315 Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present In one the WSP is PEG and the invention contemplates preparations wherein a compound is modified to include any of the forms of PEG that have been used to other such as and without limitation or Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in The PEG group may be of any convenient molecular weight and may be linear or The average weight of PEG contemplated for use in the invention ranges from about 2 kDa to about from about 5 kDa to about 50 from about 5 kDa to about f 0 in another the PEG moiety has a molecular weight from about 6 kDa to about 25 PEG groups generally attached to peptides or proteins acylation or reductive alkylation through a reactive group on the PEG moiety an or ester to a reactive group on the peptide or protein an or ester Using methods described a mixture of conjugate molecules can be and the advantage provided herein is the ability to select the proportion of conjugate to include in the if a mixture of peptides with various numbers of polymer moieties attached one or can be prepared with a predetermined proportion of A useful strategy for the methods are discussed in more detail of synthetic peptides consists of through a conjugate linkage in a peptide and a PEG each bearing a special functionality that is mutually reactive toward the The peptides can be easily prepared with conventional solid phase The peptides arc with an appropriate functional group at a The precursors are pur fied and fully characterized prior to reacting with the PEG Ligation of the peptide with PEG usually takes place in aqueous phase and be easily monitored by reverse phase analytical The PEGylated peptides by preparative and characterized by analytical amino acid analysis and laser desorption mass f Linkers Any group is whether positioned between peptide and vehicle or vehicle and its chemical structure is not since it serves primarily as a The linker is preferably made up of amino acids linked together by peptide in preferred the linker is made up of from to 20 amino acids by peptide wherein the amino acids are selected from the 20 naturally occurring amino Some of these amino acids may be as is well understood by those the In a preferred the 1 to 20 amino acids are selected from and Even more a linker is made up of a majority of amino acids that arc such as glycine and preferred linkers are polyglycines and Other specific examples of linkers ID ID and GlyProAsnGlyGly ID To explain the above for means Combinations of and Ala are also The linkers shown here are linkers within the scope of this invention may he much longer and may include other linkers arc also For alkyl linkers such as wherein s could be These alkyl linkers may further be substituted by any hindering group such as lower alkyl CI lower halogen An exemplary linker is a PEG wherein n is such that the linker has a molecular weight of to 5000 to 500 The peptide linkers may be altered to form derivatives in the same manner as described Polypeptide and Peptide production A peptide having been identified may made in transformed host cells using recombinant If the vehicle component is a the or fusion product may be expressed as To do a recombinant DNA molecule encoding the peptide is first prepared using methods well known the For sequences coding for the peptides could be excised from using suitable restriction the DNA molecule could be synthesized using chemical synthesis such as phosphoramidate a combination of these techniques could The invention therefore provides polynucleotides encoding a compound of the j The invention also provides vectors encoding compounds of the invention in an appropriate The vector comprises the polynucleotide that encodes the compound operatively linked to appropriate expression control Methods of effecting this operative either before after the polynucleotide is inserted into the are well Expression sequences include binding start stop cap polyadenylation and other signals involved with the control of transcription or The resulting vector having the polynucleotide therein is used to transform an appropriate This transformation may be performed using methods well known in the Any of a large number of available and host cells may be used in the practice of this The selection of a particular host is dependent upon a number of factors recognized by the These for compatibility with the chosen expression toxicity of the peptides encoded by the rate of of recovery of the expression and A balance of these factors must be struck with the understanding not hosts may be equally effective for the expression of a particular DNA Within these general useful microbial hosts include bacteria as yeast as and other mammalian cells in or other hosts known in the the transformed host is cultured and Host cells may be cultured under conventional fermentation conditions so that the desired compounds are Such fermentation conditions are well known in the the peptides are purified from culture by methods known in the Depending on the host utilized to express a compound of the carbohydrate groups may conveniently be attached to sites that are known to be glycosylalton sites in oligosaccharides are attached to serine or threonine residues while inked oligosaccharides are attached to asparagine residues when they are part of sequence where X can be any amino acid except X is preferably one of the 19 naturally occurring amino acids not counting The structures of and oligosaccharides and the sugar residues found in each type are One type of sugar that is commonly found on both is acid to as sialic Sialic acid is usually the terminal residue of both and oligosaccharides by virtue of its negative may confer acidic properties to the glycosylated Such may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds in mammalian cells such as such sites may further glycosylated by synthetic or synthetic procedures known in the the compounds may be made by synthetic For solid phase synthesis techniques may be Suitable techniques are well known in the and include those described in and Panayotis Mem field Davis Stewart and Young Solid Phase Peptide Finn et The Proteins and Erickson et The Proteins Solid phase synthesis is the preferred technique of making individual peptides since it is the most method of making small Compounds that contain peptides or which contain groups are particularly amendable to synthesis by organic chemistry WSP modification For obtaining a compound covalently attached to a any method described herein or otherwise known in the art Methods for preparing chemical derivatives of polypeptides or peptides will generally comprise the steps of reacting the peptide the activated molecule as a reactive ester or aldehyde derivative of the polymer under conditions whereby the polypeptide becomes attached to one or more polymer and obtaining the reaction The optimal reaction conditions be determined based on known parameters and the desired For the larger the ratio of polymer the greater the percentage of attached polymer A biologically active molecule can be linked to a polymer through any available functional group using standard methods well known in the Examples of functional groups on either the polymer or biologically active molecule which can be used to form such linkages include amine and carboxy thiol groups such as in cysteine aldehydes and and hydroxy groups as can be found in and The polymer can be activated by coupling a reactive group such as triazine el Che et or succinate et Cancer in order to react with an amine functionality on the active Another coupling method involves formation of group on one molecule and an hydrazidc or group on the other molecule to be conjugated and et Bioconjugate Ceoghegan and Bioconjugate et Other methods involve formation of an active ester at a free alcohol group of the to be conjugated using or which can then be conjugated to an amine group on the other molecule to be coupled et and 1 Patent Patent Other reactive groups which may be attached via free alcohol groups arc set forth in EP which describes the use of imidates for coupling via free Another chemistry involves acylation of the primary amines of a target using the of Acylation with results in an amide linkage which will eliminate the charge from the original Other methods utilize mild oxidation of a target under conditions selected to target the pendant diol of the penultimate glycosyl unit sialic acid for oxidation to an The resultant was then reacted with a to form a hydrazone between PEG and The hydrazone is subsequently reduced by sodium cyanoborohydride to produce a stable PEG See for Patent et July 1 specific applications of techniques for chemical for Patent states that reductive alkylation was used for attachment of polyethylene glycol molecules to an Patent discloses conjugates for enzymes and Patent discloses the modification of the number of lysine residues in proteins for the attachment polyethylene glycol molecules via reactive amine Patent discloses substantially water soluble involving for the proteins interferon and The methods of et involve the utilization of unique linkers to connect the various free amino groups in the protein to Patent and teach methods allowing for selectively chemically modified proteins and analogs including and consensus these modified proteins have advantages as relates to protein as well as providing for processing WSP modification is also described in Francis et Stability of protein in pathways of degradation and strategies for protein stabilization and is In another the method described in Delgado et of PEG to Protein By Activation With Applications immunoaffinity Cell Fisher et Separations Using Aqueous Phase Applications In Cell Biology and Plenum 1989 21 which involves the use of which results in no linkage group between the WSP moiety and the polypeptide moiety other attachment of a WSP is effected through use esters of carboxymethyl methoxy polyethylene well known in the For other descriptions of modification of a target with a for patent application EP EP EP WO Patent International application WO and O Greenwald et Crit Rev Therap Drug Carrier et Controlled Harris et Clin Zalipsky et Bioconjug 1 Nathan et et Bioconj and Francis Focus on Growth Factors i Reductive alkylation one covalent attachment of a WSP is out by reductive alkylation chemical modification procedures as provided herein to selectively modify the terminal and testing the resultant product for the desired biological such as the biological activity assays provided Reductive alkylation for attachment of a WSP to a protein or peptide exploits differential reactivity of different types of primary amino groups lysine versus the for in a particular Under the appropriate reaction substantially selective derivatizalion of the protein at the with a carbonyl group containing polymer i For reductive the selected could have a single reactive aldehyde A reactive aldehyde for polyethylene glycol which is water or mono C i alkoxy or aryloxy derivatives thereof Patent one reductive alkylation is employed to conjugate a methyl polyethylene to a Under appropriate this approach has been demonstrated to yield PEG conjugates modified through the at the An aldehyde functionality useful for conjugating the biologically active molecule can be generated from a functionality having adjacent amino and alcohol a for an threonine or hydroxyl ysine can be used to generate an aldehyde functionality oxidative cleavage under mild conditions using These or their can be normally for example at the terminus of a may be exposed via chemical or enzymatic or may be introduced via recombinant or chemical The reaction conditions for generating the aldehyde typically involve addition of a molar excess of sodium meta periodate and under mild conditions to avoid oxidation at other positions in the The pH is preferably about A typical reaction involves the addition of a 1 fold molar excess of sodium eta followed by incubation for minutes at room temperature in the The aldehyde functional group can be coupled to an activated polymer containing a hydrazide or semicarbazide functionality to form a or semicarbazonc polymers are commercially and can be if using standard PEG hydrazides for use in the invention can be obtained from Shearwater 2307 Spring Branch part of Nektar Industrial San CA The aldehyde is coupled to the polymer by mixing the solution of the two components together and heating to about until the reaction is substantially An excess of the polymer hydrazide is typically used to increase the amount of conjugate A typical reaction time is 26 Depending on thermal stability of the the reaction temperature and time can be altered to provide suitable Detailed determination of reaction conditions for both oxidation and coupling is set forth in Geoghegan and Bioconjugate Che and Using reductive the reducing agent should be stable in aqueous solution and preferably be able to reduce only the base formed in the initial process of reductive Reducing agents are selected and without sodium sodium dimethylamine trimethylamine borate and pyridine The reaction pH affects the ratio of polymer to protein to be In the reaction pH is lower than the of a target reactive a of polymer to protein will If the H is higher than the target the ratio need not be as large more reactive groups are so fewer polymer molecules are the reaction is performed in one aspect at a pH which allows one to take advantage of tire differences between the groups of the lysine residues and that of the group of the residue of the By such selective attachment of a water soluble polymer to a protein is the conjugation with the polymer takes place predominantly at the of the protein and no significant modification of other reactive such as the lysine side chain amino one methods arc provided for covalent attachment of a to a target compound and which provide a substantially homogenous preparation of protein conjugate in the absence of further extensive purification as is required using other chemical modification More if polyethylene glycol is methods described allow for production of an protein lacking possibly antigenic linkage the giycol moiety is directly coupled to the protein moiety without potentially toxic Depending on the method of WSP attachment the proportion of WSP molecules attached to the target peptide or protein molecule will as their concentrations the reaction the optimum ratio of efficiency of reaction in that there is no excess unreacted protein or is determined by the molecular weight of the WSP In when using methods that involve sped attachment and later purification of a desired the ratio may depend on the number of reactive groups amino available Purification The method of obtaining a substantially homogeneous preparation in one by purification of a predominantly single species of modified compound from a mixture of By way of a substantially homogeneous species is first separated by ion exchange chromatography to obtain material having a charge characteristic of a single species though other species having the same apparent charge may be and then the desired species is separated using size exclusion Other methods are reported and contemplated by the includes for PCT WO published May which describes a process for fractionating a mixture of adducts comprising partitioning the adducts in a aqueous biphasic one aspect of the present invention is a method for preparing a modified compound conjugate comprised of reacting a compound having more than one amino group with a water soluble polymer moiety under reducing alkylation at a pH suitable to selectively activate the group at the amino terminus of the protein moiety so that said water soluble polymer selectively attaches to said no and obtaining the reaction and particularly for a therapeutic the reaction products are separated unreacted As ascertained by peptide mapping and a preparation is provided which comprises at least PEGylated peptide in a mixture of PEGylated peptide and unreacted In other preparations are provided which comprises at least PEGylated peptide in a mixture of PEGylated peptide and unrcacted at least PEGylated peptide in a mixture of PEGylated peptide and unreacted at least PEGylated peptide in a mixture of PEGylated peptide and unreacted at least PEGylated peptide in a mixture of PEGylated peptide and unreacted and at least PEGylated peptide in a mixture of PEGylated peptide and The following examples are not intended to be limiting but only exemplary of specific embodiments the 15 Example 1 Expression Construct Assembly A encoding a TMP fusion protein comprising a murine Fc region was constructed by combining nucleotide sequences individually encoding murine Fc and a in In the first round of the murine component was amplified with PCR primers and CCGG GTA A A GGTAfi ID In a separate a polynucleotide was amplified with primers ID and ID CGTACAGGTTTACGCAAGAAAATGG ID CTAGTT ATTGCTCAGCGG ID The resulting PCR fragments were gel purified and combined in a single tube for a second round of PCR with primers I ID The PCR product from this second round of amplification was gel purified and digested with restriction enzymes and The digestion fragment was purified and ligated into the vector pAMG21 previously digested with the same This ligation mix was transformed via electroporation into E and plated LB Colonies screened via PCR and DNA A positive clone with a nucleotide sequence ID encoding the fusion protein was identified and designated Murine fusion polynucleotide ID AAAGGAGGAA TAACAT Open RF TAAGCCATGC ATTTGTACAG ATCATCTGTC 101 CCCCAAAGCC 151 CCCGAGGTCC 201 AGTTCAGCTG GTTTGTAGAT GATGTGGAGG TGCACACAGC TCAGACGCAA 251 CCCCGGGAGG AGCAGTTCAA CGCTCAGTCA GTGAACTTCC TCAATGGCAA TGCAGGGTCA 351 ACAGTGCAGC TTTCCCTGCC CCCATCGAGA AAACCATCTC CAAAACCAAA 401 GGCAGACCGA AGGCTCCACA GGTGTACACC ATTCCACCTC CCAAGGAGCA 451 CATGATAACA 501 CTGAAGACAT TACTGTGGAG TGGCAGTGGA ATGGGCAGCC AGCGGAGAAC 551 TACAAGAACA CTCAGCCCAT CATGGACACA GATGGCTCTT ACTTCGTCTA CAGC AGAGCAACTG GGAGGCAGGA 651 GTTACATGAG GGCCTGCACA ACCACCATAC CTCTCCCACT AGGTGGAGGT GGTGGTATCG TCTGCGTCAG TGGTGGAGGT GGCGGCGGAG GTATTGAGGG CGCCAATGGC CGCATAA 3 Seque TCTCGAGGATCCG CGGAAAGAAG AAGAAGAAGA AGAAAGCCCG AAAGG Murine protein sequence ID TVPEVSSVFI 51 EVMTAQTQPR 101 151 201 FTCSVLHEGJJ HSPGKGGGGG AARAGGGGGG 251 G Example 2 Fermentation of Strain 6397 Fermentation of strain 6397 was initiated by inoculation of 500 of Luria broth with a seed culture of strain in a shake When cell density reached at 600 the contents were used to inoculate a containing of complex based growth medium g 500 g 3 g sodium 40 g 20 g 5 ml Fluka 10 ml trace metals zinc chloride cobalt chloride sodium molybdate calcium chloride cupric boric acid manganese chloride sodium citrate dihydrate ml vitamins folic acid riboflavin HC1 1 niacin pantothenic acid sodium hydroxide add water to bring to The fermenter was maintained at and pH 7 with dissolved oxygen levels kept at a minimum of When the ceil density reached OD units 600 the culture was induced by the addition of homoserine At 6 hours post the broth was chilled to and the cells were harvested by centrifugation at 4550g for 60 min at The paste was then stored at 3 Protein Refolding E paste from strain 6397 expressing was dissolved in 2250 ml lysis buffer Tris 5 pH 8 and passed through a chilled microfluidizer two at The homogenate was then centrifuged at I l for 60 minutes at The supernatant was and the was resuspended in 2400 ml of water using a tissue The homogenate was then centrifuged at for 60 minutes at The supernatant was and the pellet was resuspended in 200 ml volumes of water using a tissue The homogenate was centrifuged at for 30 minutes at 4 and the supernatant was About of the pellet was resuspended in 28 ml 20 mM pH with mg hen egg white lysozyme St using a tissue grinder and incubated at for 20 Following the suspension was centrifuged at for 30 minutes at and the supernatant was The pellet was resuspended in 35 ml 8 M guanidine 50 mM Tris pH after which 350 M DTT St was added and material was incubated at 37 for 30 The solution was then centrifuged at for 30 minutes at supernatant was then transferred to L of refolding buffer mM Tris mM arginine 3 M pH 1 M mM cystamine at with gentle stirring at Example 4 Construct Purification After about 40 hours incubation at with gentle the refold solution described in Example 3 was concentrated to 500 using a tangential flow u trafi 1 apparatus with a 30 kDa cartridge followed by diafiltralion against 3 L of A mM Tris pH The concentrated material was filtered through a Whatman and loaded to an 86 ml fast flow column cm at After washing the resin with several column volumes of Buffer the protein was eluted using a 20 column volume linear gradient to B mM Tris M pH at 0 The peak fractions were and the pool was passed through a Mustang E syringe East at The filtered material was filtered a second time through a cellulose acetate filter and stored at Example 5 Protein To a cooled stirred solution of 5 in a 100 mM sodium acetate pH containing 20 mM was added a molar excess of glycol aldehyde molecular 20 The stirring of the reaction mixture was continued at the same The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR column eluted with a M phosphate buffer with M pH at After hours the SEC HPLC analysis indicated that the majority of the protein has been conjugated to At this time the reaction mixture was into a 20 mM pH The conjugates were isolated by ion exchange chromatography using a ml Hi Trap HP Q column equilibrated with a 20 mM pH reaction mixture was loaded on the column at a rate of and the unreacted MPEG aldehyde was eluted with three column volumes of the starting A linear gradient from to 20 mM pH containing M was used to the elute the Fractions collected during ion exchange chromatography separation were analyzed by HPLC SEC as described A fraction containing the and conjugates in an approximate ratio of to 1 determined by was and sterile Example 6 In vivo Testing mice River were divided groups of and injected on days 21 42 subcutaneously with either diluting agent PBS with bovine serum or diluting agent with 50 test and protein described per kg Each group was divided in half and bled from the sinus on alternate points 52 and On day mice were anesthetized with isoflurane prior to The collected blood was analyzed for a complete and differential count using an ADVIA 120 automated blood analyzer with murine software New Example 7 human initial Lvophilized Studies The human peptibody described herein in Example 7 corresponds to a dimeric form of SEQ ID wherein the human Fc is SEQ ID having initiator methionine at the The stability of was assessed by several chromatographic Exchange Exclusion and all of which were elevated Formulations ranging in concentration from to 40 were examined for both chemical and physical degradation at accelerated refrigerated and frozen stability was evaluated with to varying pH and the inclusion of mannitol or glycine as agents and sucrose as a Mannitol and sucrose were eventually chosen for further optimization after Che other candidate showed no improvement in protein was also shown to inhibit aggregation upon over a concentration range of to The following buffers were examined in screening studies over a pH and From these screening formulated in histidine buffer at pH 5 with a small amount of added was shown to be more optimal Validation of Sucrose and in the Formulation Subsequent development efforts were focused on validating the level of mannitol and in the fonnulation at a protein concentration of approximately accommodate the anticipated dosing requirements in the The effect of mannitol and in optimizing stability was demonstrated in these studies were also initiated for the purpose of anticipating manufacturing issues and Sucrose is Beneficial in Minimizing Chemical Degradation at Elevated Temperature 157 1 effect of varying sucrose and mannitol concentrations the stability of was The protein was formulated at and 2 in order to bracket the anticipated concentration range in the samples were prepared with and without The ratio of was changed by varying the amount of sucrose and adjusting the mannitol level at each sucrose concentration to maintain iso The following ratios of were examined as percent weight per 1 and Higher ratios of sucrosemiannitol are shown to minimize chemical degradation as monitored by and As is shown in the percent main peak is compared initially and after elevated te perature storage of for i 8 weeks at The greatest loss of main peak occurs in the liquid formulation formulated in lOmM sorbitol at pH followed by the formulations with and The protective effect of sucrose in minimizing chemical degradation was also observed by HPLC analysis of samples after elevated temperature storage The percent main peak from HPLC analysis of drops significantly at the low sucrose but does not appear to change meaningfully in formulations with sucrose concentrations of greater than Interpreted these results indicate that maintaining sucrose levels at or higher is critical for the stability of upon Tabic 39 Fc in buffered at pH 5 with Loss of P and Main Peak After Weeks at Formulation Time Zero 18 Weeks Time Zero Mannitol Mannitol 1 Mannitol 1 Mannitol Mannitol pH 5 Whereas in the liquid the pH 5 significant growth in the and peak the protein shows more degradation in the peak region upon analysis of samples with lower amounts of Previous work with liquid stability samples elevated temperature has shown deamidation arises from glutamine and arginine in the which contributes to the growth in the region in liquid At refrigerated chemical degradation is not observed by exchange and HPLC in the lyophilized formulation after Song term For chromatograms did not show apparent changes under varied temperatures and controlled room temperature for 6 Due to the lack of chemical degradation in the formulation over time at controlled room temperature and much of the formulation development work centered on minimizing physical aggregation associated with Minimizes Lyophilization The inclusion of at a low concentration is needed to minimize a small amount of aggregation which is apparent following This can be demonstrated by examining the relevant results from several stability studies in which samples arc evaluated for with and without the addition of A higher protein concentration of was first used to explore a wide range of in order to investigate the needed to minimize The concentration in this study was 20 with the set at and After storage for one year at aggregation is limited to in all formulations with Six month results also showed no meaningful at was chosen for further consideration in the formulation as discussed designed for at Table 40 the amount of aggregation in monitored at time 3 and 1 1 months after storage at In this was at in the aforementioned formulation and in formulations with varying mannitol ratios without In stability was followed in the current formulation without and buffered at pH 5 and Results show that only the aforementioned formulation has minimal aggregation at pH In formulations without aggregation varies from to around Aggregation is also higher at pH and compared to that detected at pH Lower ratios and 1 sucrose have higher as levels are typically around Over the level of aggregation remains consistent in the aforementioned formulation and in the formulation without through year timepoint in varied Measured Percent Aggregation Formulation Time Zero 3 Months I 1 pH pH pH pH pH pH pH 1 The optimized formulation samples were selected for evaluation at the 1 month Additional formulation studies were designed to confirm the beneficial effect of in minimizing of the samples in these studies were at pH 5 with without Table lists the percent aggregation after storage at for time intervals of 18 weeks and year in At time immediately following aggregation is minimized in all of the samples with Small amounts of aggregation are observed in samples without with the amount found in the formulation with The effectiveness of in minimizing aggregation also extends to the week with higher percent aggregation found in samples lacking and at low mannitol After storage for one year at aggregation is also consistently low in the samples 161 L containing Table 41 in varied pH 5 Measured Aggregation after at Formulation Time Zero 4 Months Year 2 4 Mannitol 2 4 Mannitol Mannitol Mannitol Mannitol Mannitol Mannitol Mannitol Mannitol Mannitol 1 Samples with were selected for evaluation at the year timcpoinl Another stability designed to test the effectiveness of antioxidants in minimizing chemical reinforced the protective effect of Antioxidants did not have an impact in minimizing chemical degradation upon elevated temperature the aforementioned with and at an T concentration of had aggregation at time zero and after 5 months storage at The same formulation without had aggregation at zero and aggregation after storage for 5 months The stability study results presented in Tables and the aforementioned studies illustrate the protective effect in minimizing aggregation upon The growth of aggregation over time is as timepoinls extending to 1 year at show no meaningful in aggregation in samples formulated with Based on these stability study results which show that the addition minimizes aggregation upon work was initiated with the recommended Studies is Concentration Dependent An initial study was designed to simulate manufacturing conditions and examine the robustness of the formulation with respect to shipping stress and stability upon was buffer exchanged into the formulation buffer using a tangential flow filtration to larger scale The protein was subsequently diluted to concentrations of and 1 with also added prior to the final filtration After samples were lyophiiization was An exemplary lyophilzation process is set forth Thermal T reatment Steps Time Step R Step H Step 60 R Step 360 H Step 60 R Step 60 H Freeze Temp Additional Freeze 0 min Condenser Setpoint C Vacuum Setpoint 100 mT Primary Drying Steps temp time Vac Step 15 H Step 100 R Step 600 H Step 600 100 H Step 800 100 R Step 25 800 R Step 25 800 100 H Step 25 800 H Step 25 100 H Step 25 H Slep 25 H Step 25 100 H Step 25 H Step 25 0 H Step 25 0 H Step 25 H Post Heat 25 100 H Secondary Temperature 28C Passive storage at resulted in more aggregation at low concentration At time aggregation was determined by to be in the 1 whereas aggregation was detected in the protein formulated at After six months storage at refrigerated the aggregation remained at the same levels as observed for the time zero samples for both concentrations of in agreement with results from previous stability Due to the higher amount of aggregation observed at the lowest concentration it was decided that would best suited as the concentration of choice for additional Aggregation Does Not Increase Upon Simulated Shear Stress samples from the initial study were also subjected to simulated ground and air transportation with aid of simulation the protocol as outlined in the ASTM Society of Testing Method was Simulated ground and air transportation was accomplished using an Blecirodynamie Vibration Table Model and a Power Model TA240 Following the transportation the physical appearance of the cakes was compared to passive with the result that no morphological changes of the cake were Both chemical and physical stability was acceptable with aggregation consistent in the stressed samples and passive controls in the samples compared to in the Stability upon reconstitution was examined in this study by preparing freshly reconstituted samples and incubating for 7 and 14 days with slow or vigorous Table 42 shows the results for and As is the amount of aggregation is minimized in the at Compared to the slow tumbling over time the no meaningful increase in aggregation is apparent over the day The amount of dimerization in these formulations and is also the shaking results appear to display a the aggregation drops to less than detectable levels after time zero in both the and there is a corresponding increase in the amount of dimerization observed in most shaken samples at each suggesting that there is some reversibility in going from the aggregate to the dimer state upon shear stressing 42 Fc in with Mannitol and pH 5 Measured Percent Aggregation and Dimerization at Zero 3 Days 7 Days 14 Days dimer dimer dimer Tumbling Shaking Tumbling Shaking Secondary for 12 Hours in the Lyophilization Cycle is Sufficient for Minimizing Residual Moisture A second study was performed in which was exchanged and diluted to in the recommended Lyophilization was achieved a cycle consisting of an initial freezing step at folio wed by at The temperature was then ramped down to held for an and primary drying initiated at with a vacuum setpoint of 1 Following a brief hold period at the temperature was ramped down to over a two hour maintained at for 20 and then gradually raised to after around 27 hours for the beginning of secondary Secondary drying was continued for a minimum of 12 hours at During the lyophilization samples were pulled after 18 and 24 hours of secondary drying in order to check the stability and compare the level of residual Results show that residual as measured by Karl fisher is around or less in ail samples Moisture is similarly low in the buffer placebo Based on this the secondary drying time of the cycle can shortened to a range between 12 43 Moisture in Secondary Drying Held at 18 24 Hours Secondary Drying Time Karl Fisher Percent Moisture 2 hours hours f Buffer Placebo I hours hours Buffer Placebo 24 hours 24 hours Buffer Placebo Stability results arc also comparable over this secondary drying time as samples do not have with to chemical or physical For the amount of aggregation is for all samples while the percent dimer is consistently at These results confirm that the secondary drying time can be shortened to less than 24 hours without affecting initial stability of the Additional work was performed to evaluate the robustness of the formulation with respect to varying excipient Since previous stability work had shown that sucrose for can have an impact on it was necessary to examine the robustness of upon minor changes in excipient Statistical Study of Robustness of Formulation An initial statistical study had been designed to examine minor changes in formulation variables such as the formulation histidine buffer strength and the using an software These samples were lyophilized but a cycle was used which resulted in higher aggregation than typically The study was a screening assuming a linear response Results of the stability assessment showed that the pH to and histidine buffer from 5 to 15 had little impact on the stability of In order to verify the contribution of and the sucrosermannitol ratio in the overall stability of a statistical design study was initiated using the more optimal lyophilization j Quadratic Statistical Stability Study The second statistical design study examined variations in and the and 2 and variations in the protein concentration and 1 The pH of the formulations were also adjusted to 5 and and the buffer strength varied from and 1 Samples were prepared and lyophilized using a Virtis with an optimized conservative cycle used for the previous stability Stability results interpreted using design software in two an assessment of the impact of formulation variables on the amount of aggregation and dimerization observed at time and the effect of formulation variables affecting Lemperature storage stability as measured by rates of change from time Zero Formulation Results from Quadratic Statistical Study Results from the time zero assessment showed as minimizes however the protein concentration was also significant in reducing the tendency to aggregate upon The software program assesses the effects that different input variables formulation have on aggregation and dimerization during With respect to dimerization at ti e not one excipient of the formulation was considered on to have a significant Several formulation impacted amount of aggregation observed upon Based on the summary results provided by concentration had the greatest effect on the degree of aggregation observed at time followed by the Aggregation is observed to be the at time in the low concentration higher amounts of have more of a protective effect in minimizing aggregation at time although this trend is as meaningful as protein concentration Higher amounts of aggregation were observed in this study compared to previous study and around aggregation was observed in some samples at Sower protein concentrations with It was observed that the variability in aggregation drops as the protein concentration At and higher concentrations has average lower aggregation than in samples formulated at or below at is Effective in upon Elevated Temperature Storage j The Statistical Design study was also used to assess changes upon elevated temperature Rates of aggregation were compared in the varied formulation conditions after days at by subtracting the elevated temperature results from the initial results and normalizing to one Rates which are negative thus refer to an apparent loss of the measured property over ti variables ing to results from were determined through peak purity and HP DC exclusion and dimer and moisture by which was correlated with Karl Fisher Titration results in some samples to verify Results from a comparison of the rates of change obtained from each assay technique show that changes in aggregation and oxidation were statistically with respect to the concentration and squared to two standard The squared term most likely arises from the quadratic nature of the study which assumes a curved response In this the squared term fits the model in addition to possibly suggesting that there is an interactive effect with itself which affects The other measured such as or the varied pH for did not exhibit any significant responses in affecting protein As is the case with the initial study previously discussed and shaken the amount of aggregation is lower high temperature The rate of change in these samples was used to make predictions based on the statistical model to protective effect of Table 44 shows predictions for the amount of aggregation based on the statistical design as the Tween concentration is raised from 0 to As is the rate of normalized to one month at is the loss of aggregate in these with the exception of in which aggregation would he predicted to The rate of loss of aggregate appears to plateau at concentrations of and suggesting that at low levels is sufficient in minimizing physical degradation The rate of growth of dimer is correspondingly similar under these conditions and was not statistically correlated with any of the formulation as previously mentioned Table 44 Statistical Quadratic Model Predictions Varied and Fc Concentration Based on Days Incubation at to one SEC prediction limits prediction limits Aggregation Oxidation 0 J The rate of change in measured oxidation at assuming that this corresponds to changes in the region of each is not statistically significant with respect to squared In this the model shown Table predicts that as the concentration is varied the rate of oxidation is consistent with the of the prediction The protein concentration affects as the rate of growth increases from to as the protein concentration drops from to keeping the constant at These results suggest that maintaining the concentration between to is desirable from a stability The amount of protein is also as the stability is worse at concentrations below Refrigerated Temperature Stability Considering the the following formulation was used for assessing the refrigerated temperature stability of in Histidine buffered at pH and The formulation was monitored for refrigerated temperature stability for a period of one Table 45 shows the results from this with results from time months and year As is the percent main peak as measured by and does not appear to decrease with The minor differences purity over time typical of the normal variation in resolution of the chromatographic columns and are also observed in the frozen The percent aggregation is consistent and does not appear to grow after one Table 45 Perceni Main Peak Concentration 3 months Year Frozen Starting Material Percent Main Peak Timcpoint Concentration 0 3 months Year Frozen Starting Material HPLC Perceni Aggregation Concentration 3 months 1 Frozen Starling Material is formulated in buffered at pH with and It has been shown that pH 5 is more optimal for and that the mannitol ratio is critical in minimizing chemical degradation upon storage at elevated temperature for this protein needed at a low concentration order to minimize the amount of aggregation which occurs as a of the lyophilization Stability studies for applications support these Statistical studies also been designed which the level of each excipient in the including the need for the protein concentration be at Refrigerated temperature stability of the recommended formulation does not show meaningful degradation after storage for one year at Example 8 binding peptide In order to determine the optimal formulation for lyophilized binding analyses were carried out that assessed binding peptide aggregation and stability at various pH excipient and protein binding peptide consists of two active polypeptide molecules linked to the of the Fc portion of an antibody The molecule is comprised of 574 amino acid residues with a total molecular weight of 1 The pi of the molecule is There are two disulfide bonds on each of the active There are a total of 20 cysteine residues throughout the most of which are oxidized in disulfide The sequence of binding peptide is as SD1AVEWESNGQPE ID Fc portion of the terminates at composes a linker The active polypeptide begins A234 and extends to the rest of the binding peptide pH screen The pH screen tested the stability of binding peptide at pH and At pH the screened buffers glutamic sodium and each at a concentration of At pH the screened buffers were histidine and both at 10 Hisilidine and Tris were also screened at pH each at Each of the buffers contained mannitol as a lyophilization caking agent and sucrose as an histidine buffer at pH was examined with and without the presence of a Tween 20 The protein was diluted to 5 with each of the formulation This solution was then dialyzed into each of the formulation buffers using dialysis tubing with a Da molecular weight exclusion where a total of exchanges were performed with a minimum of 4 hours of equilibration between After the protein was aliquoled 3 cc glass vials with a 1 ml fill These vials were then lyophilized using a drying After the vials were sealed and stored for incubation at and where individual vials were pulled and analyzed at various points over beginning immediately after lyophilization and extending out to a period of 24 The samples were reconstituted with the appropriate volume of water and analyzed for protein stability using size exclusion liquid chromatography and electrophoresis detect dimerization and proteolytic anion exchange liquid chromatography detects the properties of the cake such as reconstitution limes and moisture and properties of the reconstituted liquid solution were analyzed as Lvoohilized peptide Excipient Study The excipient screen was performed a single i 0 mM hisili ine at pH The two excipients compared in this study were arginine and 1 The caking agent used with arginine was and caking agent used with sucrose was Each of the formulations was tested at protein concentrations of 1 30 and one formulation containing a manntiol sucrose combination was tested at 30 Each of the formulations contained Tween The protein was first concentrated to 70 and dialyzed into the appropriate formulation using a The protein was then diluted to each of the three concentrations with the appropriate formulation The protein was then aliquoled into 3 cc glass with a fill volume of I The vials then lyophilized using a After the vials were sealed and stored for incubation at and where individual vials were pulled and analyzed at various points over beginning immediately after lyophiiization and extending out to a period of 24 The samples were analyzed for protein stability using size exclusion anion exchange chromatography and The properties of the cake and reconstituted liquid solutions also Lyophilizcd binding peptide Concentration 1003081 The concentration screen was performed in 10 mM histidine at pH with mannitol as the caking agent and sucrose as an The protein was concentrated to approximately and dialyzed into the formulation using a The dialyzed protein was then diluted to 30 60 and the formulation The solutions were then aliquoted into 3 cc glass vials at a fill volume of The vials were lyophilizcd using a the vials were sealed and stored for incubation at and where individual vials were pulled and analyzed at various points over beginning immediately after lyophiiization and extending out to a period of 24 The samples were analyzed for protein stability using size exclusion anion exchange chromatography and The properties of the cake and reconstituted liquid solutions were also Conclusion Considering the the optimal comprises 10 mM pH Example 9 Lyophilizcd binding peptide In order to determine the optimal formulation for lyophilizcd binding analyses were carried out that assessed binding peptide aggregation and stability at various pH excipient and protein binding peptide is a against the B cell activation factor aimed against B The peptibody is constructed of two polypeptides with a total mass The isoelectric point for this peptibody has been estimated to be pH Fc SEQUENCE fSBO ID GQ PREPQVYTtPPSRDELT the sequence of binding peptide binding peptide is as GCKWDLLllCQWVCDPLGSGSATGGSGSTASSGSGSATHMLP VSLTCLVKGPYPSDiAVEWESNGQPENNYKTTPPVLDSDGSF FIYS SRWQQGNVFSCSV HEALHNHYTQKS G K ID binding peptide broad Screen at The stability was assessed primarily by which was at elevated To assess the stability and reconstitution properties of lyophilized binding peptide over the pH range of binding peptide was formulated in various mM buffers the presence of mannitol and The following buffers were tested at approximately pH unit phosphate and For the formulation development purified bulk material was obtained in the 30 frozen liquid The material was dialyzed into the appropriate formulation buffers and lyophilized in a Virtis lyophilizer using a conservative The annealing step was performed at and lasted for 4 hours to allow for mannitol The primary drying was performed at the temperature for 20 The primary drying reached completion at since no spike in the vacuum was observed as the shelf temperature was increased up to No major collapse was observed and the proceeded successfully through the secondary drying first at and then at Upon reconstitution the formulations with pH at or above 7 were slightly while the other formulations were This was explained by the proximity of the high pH formulations to the isoelectric point of binding peptide analysis revealed a dimer as the main high molecular weight It was observed that relative dimer was strongly pH dependent with lowest accumulation at pH 5 and The amount of soluble aggregates also showed some pH No soluble aggregates were observed in the samples prior to lyophil In a small amount of dimer was present in all formulations prior and it increased further in the reconstituted post No significant clipping was observed in all The highest amount of the intact monomer was the case of succinate and histidine formulations at pH 5 and Lyophiiized binding peptide broad pH Screen at 30 stabilizing effect of sucrose and mannitol The stability was assessed primarily by which was To evaluate the effect of the presence of mannitol and sucrose on stability and reconstitution properties of lyophiiized binding peptide over pH range of binding peptide was formulated at mM histidine and phosphate Pyrophosphate and Tris buffers were excluded from this study due to poor in previous broad pH Acetate buffer was excluded due to possibility of pH changes the reconstituted samples as a result of acetate sublimation during For the formulation development purified bulk material was obtained in the 30 L frozen liquid The material was dialyzed into the appropriate formulation buffers and lyophiiized in a Virtis lyophilizer using a conservative The annealing step was performed at and lasted for 5 hours to allow for more complete mannitol The primary drying was performed at the shelf temperature of for 20 hours primary not quite reached completion at and spike in the vacuum was observed as the temperature was increased up to No major collapse was observed and the samples proceeded successfully through the secondary drying first at and then at Upon reconstitution formulations with pH around 7 were slightly while all other formulations were As it was mentioned this could be explained by the proximity of the pH formulations to the isoelectric point of binding analysis revealed dimer as the main high molecular weight it was observed that relative dimer was strongly pH dependent with the lowest accumulation at pH 5 and The amount of soluble aggregates did not show clear pH No aggregates were observed in the samples prior to dimer was observed in the bulk prior to formulating and it increased up to for high pH formulations prior to lyophilization as a result of buffer ex eh an concen trati 100322J The pH dependence of the dimer accumulation was also confirmed by close correspondence of the relative amount dimer at a given pH irrespective of the type of dimer did not increase significantly as evidenced by The exception were the samples sucrose and which showed increase in In the presence of sucrose the main peak loss was minimal even after buffer for succinate and histidine formulations with pH i and Compared to all phosphate formulations showed loss the main peak prior to the cake was formed even in the absence of both sucrose and the corresponding main peak dropped by compared to the formulations in reconstitution of formulations was much longer and required some In order to ensure robustness of the cake mannitol or glycine were included as bulking agents all subsequent formulations even though sucrose alone was shown to confer sufficient protein 3 binding peptide narrow pH Screen at 30 The stability was assessed primarily by and which were at elevated Since recovery the main peak was higher at pH 5 and the phosphate buffer was omitted the narrow pH In addition to the and histidine which performed well in the broad pH narrow pH screen included pH The formulations were tested at pH unit The sucrose and mannitol content was kept constant at and except for two succinate formulations at pH with 2 and six potential generic lyo formulations were tested at every pH such 1 20 sucrose at pH 20 sucrose at pH 20 mM sucrose at pH 20 mM sucrose at pH 20 mM sucrose at pH 20 mM sucrose at pH For the formulation development purified bulk material was obtained in the 30 liquid The material was dialyzed into the appropriate formulation buffers and lyophitized in a Virtis using a conservative Tire lyophilization cycle was The annealing step was performed at to allow for glycine and lasted for 5 The primary drying was performed initially at a short period of time the temperature was raised to and kept constant for 24 primary drying did not quite reach completion at as evidenced by a small spike in the vacuum as the shelf temperature was further increased up to no major collapse was observed and the samples proceeded successfully through the secondary first at and then at Up to 6 months lyophilized slate stability data was generated at to assess stability of binding Increase in pH results in the loss of the main peak for all histidine Only generic formulations were monitored up to 6 but the advantage of the pH was already obvious for the and data for the generic formulations suggests that formulations are more stable than especially at pH 6 and in the case of glutamate and succinate formulations there was also a clear increase in the amount of main degradation as pH becomes less Similar pH dependence is seen for the The highest recovery of the main peak was observed for pH 5 and of the main peak for can be explained by protein degradation during buffer exchange and concentrating as evidenced by a similar loss the formulations prior to stability data that glutamate formulations had higher physical stability than succinate counterparts the same has to be noted that in this study we also tested effect of increased sucrose concentrations on the stability of succinate In addition to we compared and sucrose formulations in pH The increase in sucrose decreased molecular weight to some but it did not significantly affect the amount of the main sucrose was considered optimal since such formulations most closely matched with physiological One of clip specie was observed growing at low pH by A significant portion of the dipping in the formulations occurred prior to as a result of buffer exchange and protein concentrating After lyophilization no significant increase in the amount of clips was observed even after to storage at In general the data suggests using higher pH formulations to mitigate the clipping since it is unobservable at pH 6 and the amount of the dimer is significant at higher pH and may be as high as at pH 6 a compromise can be found in formulating 3 binding peptide at pH where clippi g is especially in the glutamate and histidine and when the dimer formation is still sufficiently Conclusion mannitol and sucrose have provided sufficient cake and protein stability as confirmed by study at sucrose at pH 5 0 mM sucrose at pH can be used for formulating 30 binding In this study shows that 20 mM sucrose at pH also performs well and can be considered as a possible generic formulation for Example 10 binding peptide In order to determine the optimal formulation for lyophilized binding analyses were earned out that assessed binding peptide aggregation and stability at various pH excipient and protein binding peptide is a peptibody against the protein aimed against The peptibody is constructed of two glycosylated inked polypeptides with a total mass The isoelectric point for this peptibody has been estimated to be pH Pc SEQUENCE IS RTPEVTC V V V D S H B P E V K F N W Y V D G V E V H N A K T K P R E E Y S T Y R GQ GQPENNYKTTPPVLDSDGSFFLYSKLTVDKS S V M H E A N H Y T Q S L S S P G the sequence of binding peptide binding peptide is as MDKTHTCPPCPAPELLGGPSVFLFPPKP VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYMSTYR GQ WMCPPEGWE ID Determination of pH condition for binding peptide The stability was assessed primarily by HPLC which was at elevated A pH screen study was designed and performed to determine the optimal formulation in the liquid state prior to the Protein was formulated in pH and with agents of acetate and histidine and sucrose as the stabilizer lyo The formulated vials were stored at for up to Stability was monitored using aggregation rate constants were calculated for each of the formulation The aggregation rate at pH was found to be the therefore pH was selected as preferred formulation pH binding peptide buffer study at The stability was assessed primarily by HPLC which was A dosage form as investigated using three different buffering mM histidine and 10 succinate at pH All formulations contain For the formulation development purified hulk material was obtained in the 30 frozen liquid The material was dialyzed into the formulation buffers and lyophilized in a iyophilizer using a conservative Lyophilized protein formulation displayed acceptable cake Upon reconstitution formulations were clear Lyophilized binding peptide was stored at and The time stability studies were carried out at and found to be comparable for these formulations for up to 3 However at storage 3 the histidine containing formulation was slightly beter than the glutamate containing The succinate containing formulation was significantly less stable than the other two Based on these histidine and glutamate were considered as the preferred buffer agents for the final binding peptide j Lyophilized binding Excipient Study at 30 stabilizing effect of trehalose and starch The stability was assessed primarily by HPLC which was To evaluate the effect of the presence of starch and sucrose on stability of lyophilized binding binding peptide was formulated at in 10 glutamate buffer with The concentration of trehalose and sucrose used was I starch was added to the sucrose formulation to make a final formulation oF 10 mM 1 All formulations contain polysorbate For the formulation development purified bulk material was obtained in the 30 frozen liquid The material was dialyzed into the appropriate formulation buffers and lyophilized in a Virtis lyophilizer using a conservative Lyophilized protein formulation displayed acceptable cake Upon reconstitution formulations The stability of these formulations was monitored using Lyophilized binding peptide was stored at 37 and The real time shelf condition stability was found comparable between these I I formulalions for up to 3 However under storage condition for 3 the sucrose containing formulation was slightly better than the trehalose containing Addition of hydroxyethyl starch did not display negative impact on Based on these sucrose was considered as the preferred stabilizer for the final binding peptide Lyophilized binding peptide Excipient Study at 30 stabilizing and mannitol The stability was assessed by HPLC which was To evaluate the effect of the presence of variable amount of mannitol and sucrose on stability and reconstitution properties of lyophilized binding peptide over the mannitol range of to and sucrose range of to binding peptide was formulated at in 1 glutamate All formulations contain For the formulation development bulk material was obtained in the 30 frozen liquid The material was dialyzed into the appropriate formulation buffers and lyophilized in a Virtis using a conservative Lyophilized protein formulation displayed acceptable cake Upon reconstitution formulations were The stability of these formulations was monitored using Lyophilized binding peptide was stored at 37 and The real time shelf time condition stability was found comparable between these formulations for up to 3 However when stored at 3 an increasing amount of sucrose was found contributing to the increase in stability against Due to a concern to maintain the isotonic condition for the final formulation which limits the total amount of disaccharides and to maintain a proper ratio of mannitol and sucrose to preserve lyophilized cake mannitol and sucrose were the preferred excipients for the final Lyophilized binding peptide Excipient Study at 85 The stability was assessed primarily by HPLC which was To evaluate the effect of the protein concentration on stability insufficientOCRQuality

Claims (7)

224966/3 What is claimed is:
1. A stable lyophilized therapeutic peptibody composition comprising a buffer, a bulking agent, a stabilizing agent, and a surfactant wherein; said buffer is 10 mM histidine; the pH is 5.0; said bulking agent is 4% w/v mannitol; said stabilizing agent is 2% w/v sucrose; said surfactant is 0.004% w/v polysorbate-20; and said therapeutic peptibody is a dimer that comprises a structure of the formula Fl-(Ll)e-Pl-(L2)f-P2 wherein: F is an Fc domain, wherein the Fc domain is attached at the N-terminus of -L -P -L -P ; P 1 and P 2 are each independently selected from Table 6; L and L are each independently linkers; and e and f are each independently 0 or 1.
2. The composition of claim 1 wherein P 1 and P 2 have the same amino acid sequence.
3. The composition of claim 1 wherein the Fc domain is set out in SEQ ID NO: 1.
4. The composition of claim 1 wherein the therapeutic peptibody concentration is between about 0.25 mg/mL and 250 mg/mL.
5. The composition of claim 1 wherein the therapeutic peptibody concentration is 0.5 mg/mL.
6. A kit for preparing an aqueous pharmaceutical composition comprising a first container having a stable lyophilized therapeutic peptibody composition of claim 1, and a second container having a physiologically acceptable solvent for the lyophilized composition.
7. The composition of claim 1, wherein the Fc domain is SEQ ID NO: 1 and wherein the
IL224966A 2006-04-21 2013-02-27 Lyophilized therapeutic peptibody formulations IL224966A (en)

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Families Citing this family (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100719202B1 (en) 1998-10-23 2007-05-16 키린-암젠 인코포레이티드 A COMPOUND BINDING TO MPl RECEPTOR AND A PHARMACEUTICAL COMPOSITION THEREOF
US7658938B2 (en) 1999-02-22 2010-02-09 Merrion Reasearch III Limited Solid oral dosage form containing an enhancer
CA2634663C (en) 1999-02-22 2009-08-25 University Of Connecticut Novel albumin-free factor viii formulations
ES2529706T3 (en) 1999-06-01 2015-02-24 Biogen Idec Ma Inc. A blocking monoclonal antibody against the alpha1-I domain of VLA-1, and its use for the treatment of inflammatory disorders
WO2002083854A2 (en) 2001-04-13 2002-10-24 Biogen, Inc. Antibodies to vla-1
DK1994155T4 (en) 2006-02-13 2022-07-25 Daiichi Sankyo Co Ltd POLYNUCLEOTIDE AND POLYPEPTIDE SEQUENCES INVOLVED IN THE BONE MODELING PROCESS
US8168181B2 (en) 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
AU2007235251B2 (en) 2006-04-07 2013-02-07 Merrion Research Iii Limited Solid oral dosage form containing an enhancer
US9283260B2 (en) * 2006-04-21 2016-03-15 Amgen Inc. Lyophilized therapeutic peptibody formulations
AU2011265555B2 (en) * 2006-04-21 2016-03-10 Amgen Inc. Lyophilized therapeutic peptibody formulations
DK2034830T3 (en) 2006-05-25 2014-10-27 Biogen Idec Inc ANTI-VLA-1 ANTIBODY FOR TREATMENT OF CASES
AU2009244799B2 (en) 2008-05-07 2014-11-20 Merrion Research Iii Limited Compositions of GnRH related compounds and processes of preparation
JP5779780B2 (en) 2008-11-07 2015-09-16 バクスター・インターナショナル・インコーポレイテッドＢａｘｔｅｒ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃｏｒｐ０Ｒａｔｅｄ Factor VIII formulation
WO2011038139A1 (en) 2009-09-23 2011-03-31 Amgen Inc. Treatment of ovarian cancer using a specific binding agent of human angiopoietin-2 in combination with a taxane
MX2012008453A (en) * 2010-01-19 2012-11-21 Hanmi Science Co Ltd Liquid formulations for long-acting erythropoietin conjugate.
US9089484B2 (en) 2010-03-26 2015-07-28 Merrion Research Iii Limited Pharmaceutical compositions of selective factor Xa inhibitors for oral administration
DK2646052T3 (en) 2010-12-02 2017-07-17 Oncolytics Biotech Inc LYOPHILIZED VIRAL FORMULATIONS
EA201390810A1 (en) 2010-12-02 2013-09-30 Онколитикс Байотек Инк. LIQUID VIRAL COMPOSITIONS
JP5759211B2 (en) * 2011-03-11 2015-08-05 三洋化成工業株式会社 Freeze drying method
EP2726088B1 (en) 2011-06-29 2019-01-02 Amgen Inc. Predictive biomarker of survival in the treatment of renal cell carcinoma
DE102011055889B4 (en) * 2011-11-30 2013-08-01 Otc Gmbh Process for the preparation of a protein hydrolyzate
CN102586217A (en) * 2011-12-16 2012-07-18 深圳市海普瑞药业股份有限公司 Freeze-drying preservation method for heparin flavobacterium heparinum heparinase I, II and III
RU2482128C1 (en) * 2011-12-28 2013-05-20 Замертон Холдингс Лимитед Peptides possessing cytoprotective activity
CN104271122A (en) 2012-02-16 2015-01-07 桑塔鲁斯股份有限公司 Anti-vla1 (cd49a) antibody pharmaceutical compositions
EP2644186A1 (en) 2012-03-26 2013-10-02 OTC GmbH Hair conditioning composition for permanent and semi-permanent hair coloration applications
WO2014004578A1 (en) * 2012-06-25 2014-01-03 Emergent Product Development Gaithersburg Inc. Temperature stable vaccine formulations
ES2723885T3 (en) 2012-07-19 2019-09-03 Daiichi Sankyo Co Ltd Anti-Siglec-15 antibodies
EP2698162A1 (en) * 2012-08-15 2014-02-19 Credentis AG Method for producing a composition for treating a tooth lesion
CN105530949A (en) * 2013-03-15 2016-04-27 安姆根有限公司 Myostatin antagonism in human subjects
CN104274827B (en) * 2013-07-01 2020-07-14 上海贺普药业股份有限公司 Formulations of He Pula peptide
WO2015035405A1 (en) * 2013-09-09 2015-03-12 Pinta Biotherapeutics, Inc. Myostatin antagonist for treatment of pew in esrd patients
EP3065756B1 (en) * 2013-11-07 2019-02-20 Dr. August Wolff GmbH & Co. KG Arzneimittel Storage stable lyophilized tripeptide formulations
US10801070B2 (en) 2013-11-25 2020-10-13 The Broad Institute, Inc. Compositions and methods for diagnosing, evaluating and treating cancer
US11725237B2 (en) 2013-12-05 2023-08-15 The Broad Institute Inc. Polymorphic gene typing and somatic change detection using sequencing data
CN106132432A (en) * 2013-12-06 2016-11-16 博德研究所 Preparation for neoplasia vaccine
WO2015095811A2 (en) 2013-12-20 2015-06-25 The Board Institute Inc. Combination therapy with neoantigen vaccine
US10357559B2 (en) 2013-12-27 2019-07-23 Emergent Product Development Gaithersburg Inc. Temperature stable vaccine formulations
US11299528B2 (en) 2014-03-11 2022-04-12 D&D Pharmatech Inc. Long acting TRAIL receptor agonists for treatment of autoimmune diseases
CA2943919A1 (en) 2014-03-29 2015-10-08 Intas Pharmaceuticals Ltd. Lyophilized pharmaceutical composition of fc-peptide fusion protein
DK3149048T3 (en) * 2014-05-28 2020-04-14 Nono Inc Chloridsalt af tat-nr2b9c
TW201625675A (en) * 2014-06-13 2016-07-16 聖塔瑪麗亞生物治療公司 Formulated receptor polypeptides and related methods
EP3750556A1 (en) * 2014-08-20 2020-12-16 Portola Pharmaceuticals, Inc. Lyophilized formulations for factor xa antidote
JP6671141B2 (en) * 2014-10-21 2020-03-25 大日本住友製薬株式会社 Suspension
US10993997B2 (en) 2014-12-19 2021-05-04 The Broad Institute, Inc. Methods for profiling the t cell repertoire
EP3234193B1 (en) 2014-12-19 2020-07-15 Massachusetts Institute of Technology Molecular biomarkers for cancer immunotherapy
WO2016120378A1 (en) 2015-01-29 2016-08-04 Novo Nordisk A/S Tablets comprising glp-1 agonist and enteric coating
US10857229B2 (en) 2015-04-30 2020-12-08 Amgen Inc. Treatment of ovarian cancer in patients with ascites using a specific binding agent of human angiopoietin-2 in combination with a taxane
IL302102A (en) 2015-05-20 2023-06-01 Dana Farber Cancer Inst Inc Shared neoantigens
JP6797203B2 (en) 2015-12-17 2020-12-09 ザ・ジョンズ・ホプキンス・ユニバーシティー Improvement of systemic sclerosis by death receptor agonist
JP6992262B2 (en) * 2016-03-31 2022-02-15 東ソー株式会社 Manufacturing method of denaturing antibody measurement reagent
US11084879B2 (en) 2016-04-07 2021-08-10 The Johns Hopkins University Compositions and methods for treating pancreatitis and pain with death receptor agonists
GB201608323D0 (en) 2016-05-12 2016-06-29 Ucb Biopharma Sprl Pharmaceutical compositions
CN109310756B (en) * 2016-05-13 2022-06-28 奥美药业有限公司 Novel angiopoietin 2, VEGF bispecific antagonists
WO2018140391A1 (en) 2017-01-24 2018-08-02 The Broad Institute, Inc. Compositions and methods for detecting a mutant variant of a polynucleotide
JP2017155046A (en) * 2017-04-05 2017-09-07 イーストポンド・ラボラトリーズ・リミテッド Cell hydration composition comprising cyclodextrin
LT3681483T (en) 2017-09-15 2022-09-12 Amgen Inc. Process for lyophilized pharmaceutical formulation of a therapeutic protein
WO2019055902A1 (en) * 2017-09-18 2019-03-21 Amgen Inc. Vegfr-fc fusion protein formulations
CN108159413B (en) * 2018-01-10 2021-07-06 杭州洪桥中科基因技术有限公司 Freeze-dried live vaccine for animals and preparation method thereof
GB201804835D0 (en) * 2018-03-26 2018-05-09 Ge Healthcare As Formulation and method of preparation
JP7412409B2 (en) * 2018-07-31 2024-01-12 アムジェン インコーポレイテッド Pharmaceutical formulations of masked antibodies
CN113474360A (en) 2019-02-18 2021-10-01 伊莱利利公司 Therapeutic antibody formulations
EP3736574A1 (en) * 2019-05-07 2020-11-11 Atlas Antibodies AB A formulation comprising an isotope labeled fusion polypeptide
DK3744319T3 (en) * 2019-05-28 2023-01-16 Ilkogen Ilac Sanayi Ve Ticaret A S STABLE LYOPHILIZED FORMULATION FOR HYBRID FC-FUSIONED G-CSF
JP2019178154A (en) * 2019-06-18 2019-10-17 イーストポンド・ラボラトリーズ・リミテッド Cell hydration composition comprising cyclodextrin
US11767353B2 (en) 2020-06-05 2023-09-26 Theraly Fibrosis, Inc. Trail compositions with reduced immunogenicity
CN117255809A (en) * 2021-03-31 2023-12-19 杭州嘉因生物科技有限公司 Fusion molecules targeting VEGF and angiogenin and uses thereof
AR127813A1 (en) 2021-12-01 2024-02-28 UCB Biopharma SRL PHARMACEUTICAL FORMULATIONS COMPRISING Fab-PEG
US20240076597A1 (en) * 2022-09-02 2024-03-07 Terumo Bct, Inc. Methods for cell expansion, differentiation, and/or harvesting of natural killer cells using hollow-fiber membranes
CN117186173B (en) * 2023-09-14 2024-04-05 海南大学 Inhibition of Abeta 42 Tripeptide compounds that aggregate and reduce their cytotoxic effects

Family Cites Families (110)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3691016A (en) * 1970-04-17 1972-09-12 Monsanto Co Process for the preparation of insoluble enzymes
CA1023287A (en) * 1972-12-08 1977-12-27 Boehringer Mannheim G.M.B.H. Process for the preparation of carrier-bound proteins
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US3941763A (en) * 1975-03-28 1976-03-02 American Home Products Corporation PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
US4002531A (en) * 1976-01-22 1977-01-11 Pierce Chemical Company Modifying enzymes with polyethylene glycol and product produced thereby
US4195128A (en) * 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4330440A (en) * 1977-02-08 1982-05-18 Development Finance Corporation Of New Zealand Activated matrix and method of activation
CA1093991A (en) * 1977-02-17 1981-01-20 Hideo Hirohara Enzyme immobilization with pullulan gel
US4229537A (en) * 1978-02-09 1980-10-21 New York University Preparation of trichloro-s-triazine activated supports for coupling ligands
DE3244265A1 (en) 1982-11-30 1984-05-30 Cassella Ag, 6000 Frankfurt SOFTENER EMULSION, METHOD FOR THEIR PRODUCTION AND THEIR USE
US4503235A (en) 1983-03-11 1985-03-05 Warner-Lambert Company Process for producing 4-carbamoyl-1H-imidazolium-5-olate
DE3572982D1 (en) 1984-03-06 1989-10-19 Takeda Chemical Industries Ltd Chemically modified lymphokine and production thereof
DE3889853D1 (en) 1987-11-05 1994-07-07 Hybritech Inc Polysaccharide-modified immunoglobulins with reduced immunogenic potential or improved pharmacokinetics.
US4904584A (en) * 1987-12-23 1990-02-27 Genetics Institute, Inc. Site-specific homogeneous modification of polypeptides
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
GB8824591D0 (en) 1988-10-20 1988-11-23 Royal Free Hosp School Med Fractionation process
ATE135370T1 (en) 1988-12-22 1996-03-15 Kirin Amgen Inc CHEMICALLY MODIFIED GRANULOCYTE COLONY EXCITING FACTOR
US5089261A (en) * 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5225538A (en) * 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
US5627262A (en) 1989-07-05 1997-05-06 The Board Of Regents Of The University Of Oklahoma Method and composition for the treatment of septic shock
ATE194384T1 (en) 1989-09-12 2000-07-15 Hoffmann La Roche TNF-BINDING PROTEINS
JPH04218000A (en) 1990-02-13 1992-08-07 Kirin Amgen Inc Modified polypeptide
US5723286A (en) * 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems
US5252714A (en) * 1990-11-28 1993-10-12 The University Of Alabama In Huntsville Preparation and use of polyethylene glycol propionaldehyde
CA2288429C (en) 1991-03-15 2006-04-25 Synergen, Inc. Pegylation of polypeptides
US5595732A (en) * 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US5281698A (en) * 1991-07-23 1994-01-25 Cetus Oncology Corporation Preparation of an activated polymer ester for protein conjugation
US5362852A (en) * 1991-09-27 1994-11-08 Pfizer Inc. Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties
US5733731A (en) * 1991-10-16 1998-03-31 Affymax Technologies N.V. Peptide library and screening method
US5270170A (en) * 1991-10-16 1993-12-14 Affymax Technologies N.V. Peptide library and screening method
NZ244778A (en) 1991-10-21 1994-03-25 Ortho Pharma Corp Peg imidates and protein derivatives thereof
EP0615451B1 (en) 1992-05-26 2005-12-07 Immunex Corporation Novel cytokine that binds cd30
DE69332485T2 (en) 1992-08-11 2003-11-13 Harvard College Immunomodulatory peptides
GB9225448D0 (en) 1992-12-04 1993-01-27 Erba Carlo Spa Improved synthesis of polymer bioactive conjugates
US5922545A (en) * 1993-10-29 1999-07-13 Affymax Technologies N.V. In vitro peptide and antibody display libraries
US5773569A (en) * 1993-11-19 1998-06-30 Affymax Technologies N.V. Compounds and peptides that bind to the erythropoietin receptor
US5981478A (en) 1993-11-24 1999-11-09 La Jolla Cancer Research Foundation Integrin-binding peptides
US5786331A (en) * 1994-02-02 1998-07-28 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5880096A (en) * 1994-02-02 1999-03-09 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5608035A (en) * 1994-02-02 1997-03-04 Affymax Technologies N.V. Peptides and compounds that bind to the IL-1 receptor
US5844929A (en) * 1994-02-24 1998-12-01 British Telecommunications Public Limited Company Optical device with composite passive and tapered active waveguide regions
US6309853B1 (en) 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
US20030053982A1 (en) * 1994-09-26 2003-03-20 Kinstler Olaf B. N-terminally chemically modified protein compositions and methods
US5824784A (en) * 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
AU693478B2 (en) 1994-11-10 1998-07-02 Metabolic Pharmaceuticals Limited Treatment of obesity
IL116026A (en) 1994-11-22 2005-08-31 Rhone Poulenc Rorer Sa Peptides capable of linking to the sh3 domain of gap, nucleotide sequences encoding the same, their preparation and uses
US5888763A (en) 1994-12-30 1999-03-30 The Rockefeller University Peptides specific for the first Crk-SH3 domain
WO1996024369A1 (en) 1995-02-06 1996-08-15 Genetics Institute, Inc. Formulations for il-12
IL113159A0 (en) 1995-03-28 1995-06-29 Yeda Res & Dev Synthetic peptides and pharmaceutical compositions comprising them
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
US5739277A (en) * 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5869451A (en) * 1995-06-07 1999-02-09 Glaxo Group Limited Peptides and compounds that bind to a receptor
US5767078A (en) 1995-06-07 1998-06-16 Johnson; Dana L. Agonist peptide dimers
PT1516628E (en) 1995-07-27 2013-09-24 Genentech Inc Stable isotonic lyophilized protein formulation
US5746516A (en) 1995-08-11 1998-05-05 Hitachi Powdered Metals Co., Ltd. Porous bearing system having internal grooves and electric motor provided with the same
US5817750A (en) 1995-08-28 1998-10-06 La Jolla Cancer Research Foundation Structural mimics of RGD-binding sites
JPH09151200A (en) 1995-09-29 1997-06-10 Ajinomoto Co Inc Peptide capable of inducing immune response to human gastric cancer, therapeutic and preventive agent for human gastric cancer containing the same
US6369027B1 (en) 1995-12-22 2002-04-09 Amgen Inc. Osteoprotegerin
US5714577A (en) 1996-01-26 1998-02-03 University Of Pittsburgh Antimicrobial peptides
IL117223A0 (en) 1996-02-22 1996-06-18 Yeda Res & Dev Antipathogenic polypeptides and compositions comprising them
JP4046354B2 (en) 1996-03-18 2008-02-13 ボード オブ リージェンツ，ザ ユニバーシティ オブ テキサス システム Immunoglobulin-like domain with increased half-life
JP4088344B2 (en) 1996-03-28 2008-05-21 カイロン コーポレイション Peptide ligand of urokinase receptor
IL118003A0 (en) 1996-04-23 1996-08-04 Yeda Res & Dev Novel vip fragments and pharmaceutical compositions comprising them
FR2748028B1 (en) 1996-04-30 1998-08-14 Lab Francais Du Fractionnement PEPTIDES DERIVED FROM THE VON WILLEBRAND FACTOR AND THEIR USE AS ANTICOAGULANTS
WO1997046668A1 (en) 1996-06-07 1997-12-11 Takeda Chemical Industries, Ltd. Novel peptide, process for the production of the same, and use of the same
US6126939A (en) 1996-09-03 2000-10-03 Yeda Research And Development Co. Ltd. Anti-inflammatory dipeptide and pharmaceutical composition thereof
DE69734887T2 (en) 1996-09-10 2006-08-24 The Burnham Institute, La Jolla TUMOR FINDING MOLECULES, DIVIDING CONJUGATES, AND METHOD FOR USE THEREOF
KR100408229B1 (en) * 1996-10-04 2003-12-01 암겐 인코포레이티드 Pharmaceutical compositions containing an mpl ligand
JP2001512560A (en) 1996-10-08 2001-08-21 ユー―ビスイス ベスローテン フェンノートシャップ Methods and means for the selection of peptides and proteins with specific affinity for a target
WO1998033812A1 (en) 1997-02-05 1998-08-06 Brigham And Women's Hospital, Inc. Mast cell protease peptide inhibitors
IL132304A0 (en) 1997-04-16 2001-03-19 Amgen Inc Osteoprotegerin binding proteins and receptors
US6265535B1 (en) 1997-05-30 2001-07-24 The Trustees Of The University Of Pennsylvania Peptides and peptide analogues designed from binding sites of tumor necrosis factor receptor superfamily and their uses
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
DE69812727D1 (en) 1997-11-07 2003-04-30 Conjuchem Inc Opioid conjugates with endogenous carrier proteins
WO1999038526A1 (en) 1998-01-29 1999-08-05 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variant peptide ligands that selectively induce apoptosis
WO1999045026A1 (en) 1998-03-05 1999-09-10 Chiron Corporation Method for increasing the serum half-life of a biologically active molecule
US6235872B1 (en) 1998-03-12 2001-05-22 The Burnham Institute Proapoptotic peptides dependence polypeptides and methods of use
WO1999047151A1 (en) 1998-03-20 1999-09-23 Chugai Pharmaceutical Co., Ltd. Peptide ligands for the erythropoietin receptor
EP0947524A1 (en) 1998-03-30 1999-10-06 Upither B.V. Novel peptides for the treatment of autoimmune diseases
AU744044B2 (en) 1998-04-06 2002-02-14 Advanced Immunit, Inc. Short peptides for treatment of neurological degenerative diseases
AU760381B2 (en) * 1998-04-28 2003-05-15 Laboratoires Serono Sa PEG-LHRH analog conjugates
EP0972780A1 (en) 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Il-6 antagonist peptides
PL200471B1 (en) 1998-05-22 2009-01-30 Abbott Lab Peptide antiangiogenic drugs
US5932548A (en) 1998-06-03 1999-08-03 Deghenghi; Romano Lysine containing peptides for treatment of heart disease
WO2000001402A1 (en) 1998-07-02 2000-01-13 Envision Biomedical Consulting Antiproliferative and antiviral proteins and peptides
US6168785B1 (en) 1998-07-16 2001-01-02 Institut Pasteur Biological applications of new peptides of IL-2 and derivatives and use as therapeutic agents
WO2000011028A2 (en) 1998-08-21 2000-03-02 Yeda Research And Development Co. Ltd. Anti-inflammatory peptides derived from il-2 and analogues thereof
TR200101087T2 (en) 1998-10-16 2001-09-21 Biogen, Inc. Interferon beta-fusion proteins and their use
KR100719202B1 (en) 1998-10-23 2007-05-16 키린-암젠 인코포레이티드 A COMPOUND BINDING TO MPl RECEPTOR AND A PHARMACEUTICAL COMPOSITION THEREOF
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2000047740A2 (en) 1999-02-12 2000-08-17 Amgen Inc. Tnf-related proteins
US6635646B1 (en) * 1999-05-04 2003-10-21 Schering Corporation Pegylated interferon alfa-CCR5 antagonist combination HIV therapy
US6586398B1 (en) * 2000-04-07 2003-07-01 Amgen, Inc. Chemically modified novel erythropoietin stimulating protein compositions and methods
EP1278778A2 (en) 2000-05-03 2003-01-29 Amgen Inc., Modified peptides, comprising an fc domain, as therapeutic agents
US6853809B2 (en) 2001-01-30 2005-02-08 Koninklijke Philips Electronics N.V. Camera system for providing instant switching between wide angle and full resolution views of a subject
BR0209546A (en) * 2001-05-11 2004-06-29 Amgen Inc Composition of matter, DNA, expression vector, host cell, and methods for treating B-cell mediated autoimmune disease, lupus, B-cell mediated cancer, and B-cell lymphoma
RU2180233C1 (en) 2001-06-26 2002-03-10 Общество с ограниченной ответственностью "Протеиновый контур" Method of recombinant protein liquid medicinal form preparing
US7138370B2 (en) * 2001-10-11 2006-11-21 Amgen Inc. Specific binding agents of human angiopoietin-2
US7332474B2 (en) * 2001-10-11 2008-02-19 Amgen Inc. Peptides and related compounds having thrombopoietic activity
US7205275B2 (en) 2001-10-11 2007-04-17 Amgen Inc. Methods of treatment using specific binding agents of human angiopoietin-2
US20040247588A1 (en) 2002-08-28 2004-12-09 Johnson Robert E. Formulations of modified antibodies and methods of making the same
US6919426B2 (en) 2002-09-19 2005-07-19 Amgen Inc. Peptides and related molecules that modulate nerve growth factor activity
AU2003291689A1 (en) 2002-10-31 2004-05-25 Protein Design Labs, Inc. Stable liquid pharmaceutical formulation of antibodies that are prone to isomerization
AR042545A1 (en) * 2002-12-20 2005-06-22 Amgen Inc BINDING AGENTS THAT INHIBIT MIOSTATINE
US8143380B2 (en) 2004-07-08 2012-03-27 Amgen Inc. Therapeutic peptides
AU2005289685B2 (en) * 2004-09-24 2009-07-16 Amgen Inc. Modified Fc molecules
US9283260B2 (en) * 2006-04-21 2016-03-15 Amgen Inc. Lyophilized therapeutic peptibody formulations
EP3035305B1 (en) 2014-12-18 2016-11-16 Axis AB Enclosure and arrangement for recess mounting of a camera or camera head

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